JP2015519065A - Recombinant metabolic microorganism and use thereof - Google Patents

Abstract

Terpenes are effective commercial products used in a wide variety of industries. Terpenes may be produced from petrochemical sources or from terpene feedstocks such as turpentine. However, these production methods are expensive, not sustainable, and often cause environmental problems including problems due to climate change. Microbial fermentation offers an alternative option for the production of terpenes. One or more terpenes and / or precursors can be produced by microbial fermentation of a substrate containing CO. Recombinant microorganisms may be used in such methods. Carbon-oxide-nutrient acetic acid-producing recombinant microorganisms can be used in such methods. The recombinant microorganism may include, for example, an exogenous mevalonate (MVA) pathway enzyme and / or a DXS pathway enzyme. [Selection] Figure 1

Description

  The present invention relates to recombinant microorganisms and methods for the production of terpenes and / or precursors thereof by microbial fermentation of substrates containing CO.

  Terpenes are naturally occurring chemical substrates composed of isoprene units having 5 carbons and are divided into various types. Terpene derivatives include terpenoids (also known as isoprenoids) that can be formed by oxidation or rearrangement of the carbon skeleton, or addition or rearrangement of multiple functional groups.

  Examples of terpenes include isoprene (C5 hemiterpenes), farnesene (C15 sesquiterpenes), artemisinin (C15 sesquiterpenes), citral (C10 monoterpenes), carotenoids (C40 tetraterpenes), menthol (C10 mono) Terpenes), camphor (C10 monoterpenes), and cannabinoids.

  Terpenes are valuable commercial products used in a wide variety of industries. Terpenes are used in the most ton units as resins, solvents, perfumes, and vitamins. For example, isoprene is used, for example, in the production of synthetic rubber for the tire industry (cis-1,4-polyisoprene); farnesene is used as an energy-enriched drop-in fuel or jet fuel used in transportation Artemisinin is used in malaria drugs; and citral, carotenoids, menthol, camphor, and cannabinoids are used in the manufacture of pharmaceuticals, butadiene, and aroma components.

  Terpenes may be produced from petrochemical sources or from terpene feedstocks such as turpentine. For example, isoprene is produced petrochemically as a byproduct of naphtha or ethylene production petroleum cracking. Many terpenes are also extracted in relatively small amounts from natural sources. However, these production methods are expensive, are not sustainable and often cause environmental problems including contributions to climate change.

  Due to the very strong flammable nature of isoprene, known production methods require extensive safety measures to limit the possibility of combustion and explosion.

  The object of the present invention is to overcome one or more of the disadvantages of the prior art or at least provide the public with alternative means for producing terpenes and other related products.

  Microbial fermentation offers an alternative option for the production of terpenes. Terpenes are ubiquitous in nature, for example involved in bacterial cell wall biosynthesis and are produced by some trees (eg poplar) to protect leaves from UV light exposure. However, not all bacteria contain the cellular machinery necessary to produce terpenes and / or terpene precursors as metabolites. For example, C.I. autoethanogenum or C.I. Carbon oxytrophic acetic acid producers such as ljungdahlii can ferment a substrate containing carbon monoxide to produce products such as ethanol, but do not use terpenes and / or precursors of terpenes as metabolites at all. It is known that it does not produce and emit. Furthermore, most bacteria are known not to produce commercially effective terpenes.

  The present invention particularly provides a method for producing one or more terpenes and / or precursors thereof by microbial fermentation of a substrate comprising CO, and a recombinant microorganism for use in such a method.

  In a first aspect, the present invention relates to a recombinant carbon dioxide producing acetic acid-trophic acetic acid capable of producing one or more terpenes and / or precursors thereof and optionally one or more other products by fermentation of a substrate comprising CO. I will provide a.

  In one particular embodiment, the microorganism expresses one or more enzymes (referred to herein as exogenous enzymes) in the mevalonate (MVA) pathway that are not present in the parental microorganism that results in the recombinant microorganism. Adapted to do. In another embodiment, the microorganism is adapted to overexpress one or more enzymes (endogenous enzymes herein) in the mevalonate (MVA) pathway present in the parental microorganism that results in the recombinant microorganism. The

In a further embodiment, the microorganism is
(A) expresses one or more exogenous enzymes in the mevalonate (MVA) pathway and / or overexpresses one or more endogenous enzymes in the mevalonate (MVA) pathway; and (b) the DXS pathway It is adapted to express one or more exogenous enzymes in and / or to overexpress one or more endogenous enzymes in the DXS pathway.

In one embodiment, the one or more enzymes from the mevalonic acid (MVA) pathway are
a) Thiolase (EC 2.3.1.9)
b) HMG-CoA synthase (EC 2.3.3.10)
c) HMG-CoA reductase (EC 1.1.1.88)
d) Mevalonate kinase (EC 2.7.1.36)
e) Phosphomevalonate kinase (EC 2.7.4.2)
f) selected from the group consisting of mevalonate diphosphodecarboxylase (EC 4.1.1.33), and g) any variant thereof that is functionally equivalent.

In a further embodiment, the one or more enzymes from the DXS pathway are
a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7),
b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267),
c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60),
d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148),
e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12),
f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.1.7.7.1),
g) selected from the group consisting of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2), and h) any variant thereof that is functionally equivalent Is done.

In a further embodiment, one or more additional exogenous or endogenous enzymes are expressed or overexpressed resulting in the production of a terpene compound or precursor thereof, wherein the expressed exogenous or overexpressed endogenous enzyme is ,
a) geranyltransferase (EC: 2.5.1.10),
b) heptaprenyl diphosphate synthase (EC: 2.5.1.10),
c) Octaprenyl-diphosphate synthase (EC: 2.5.1.90),
d) isoprene synthase (EC 4.2.27),
e) Isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2),
f) selected from the group consisting of farnesene synthase (EC 4.2.346 / EC 4.2.3.47), and g) any variant thereof that is functionally equivalent.

  In one embodiment, the parental microorganism can ferment a substrate containing CO to produce acetyl-CoA but cannot convert acetyl-CoA to mevalonate or isopentenyl pyrophosphate (IPP), and the recombinant microorganism enters the mevalonate pathway. Adapted to express one or more enzymes involved.

  In one embodiment, the one or more terpenes and / or precursors thereof are mevalonic acid, IPP, dimethylallyl diphosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene. Selected from.

  In one embodiment, the microorganism comprises one or more exogenous nucleic acids adapted to increase the expression of one or more endogenous nucleic acids, and the one or more endogenous nucleic acids are the enzymes previously described herein. Code one or more of.

  In one embodiment, one or more exogenous nucleic acid adapted to increase expression is a regulatory element. In one embodiment, the regulatory element is a promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is selected from the group comprising the Wood-Ljungdahl gene cluster or the phosphotransacetylase / acetate kinase operon promoter.

  In one embodiment, the microorganism is adapted to encode and express one or more of the aforementioned enzymes and includes one or more exogenous nucleic acids. In one embodiment, the microorganism is adapted to encode and express at least two of the enzymes and includes one or more exogenous nucleic acids. In other embodiments, the microorganism is adapted to encode and express at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more of the enzymes. Contains one or more exogenous nucleic acids.

  In one embodiment, the one or more exogenous nucleic acids is a nucleic acid construct or vector, and in one particular embodiment is a plasmid that encodes one or more of the above enzymes in any combination.

  In one embodiment, the exogenous nucleic acid is an expression plasmid.

  In one particular embodiment, the parental microorganism is selected from the group of oxytrophic acetic acid producing bacteria. In certain embodiments, the microorganism, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium lim Selected from the group consisting of osum, Moorella thermoacetica, Moorella thermoautotropica, Sporumusa ovata, Sporomusa sylvaetica, Sporomusa sphaeroides, Oxobacter pfennigi.

  In one embodiment, the parental microorganism is Clostridium autoethanogenum or Clostridium ljungdahlii. In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693. In another specific embodiment, the microorganism is Clostridium ljungdahlii DSM 13528 (or ATCC 55383).

In one embodiment, the parental microorganism lacks one or more genes in the DXS pathway and / or the mevalonic acid (MVA) pathway. In one embodiment, the parental microorganism is
a) thiolase (EC 2.3.1.9),
b) HMG-CoA synthase (EC 2.3.3.10),
c) HMG-CoA reductase (EC 1.1.1.88),
d) Mevalonate kinase (EC 2.7.1.36),
e) phosphomevalonate kinase (EC 2.7.4.2),
f) diphosphomevalonate decarboxylase (EC 4.1.1.33),
g) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7),
h) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267),
i) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60),
j) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148),
k) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12),
l) 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1),
m) selected from the group consisting of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2), and n) any variant thereof that is functionally equivalent It lacks one or more genes encoding the enzyme to be processed.

  In a second aspect, the present invention allows one or more terpenes and / or precursors thereof to be produced by fermentation of a substrate comprising CO when expressed in the microorganism. Nucleic acids encoding the enzymes are provided.

  In one embodiment, the nucleic acid is one or more enzymes that, when expressed in the microorganism, allow the microorganism to produce one or more terpenes and / or precursors thereof by fermentation of a substrate comprising CO. Code. In one embodiment, the nucleic acids of the invention encode at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more of such enzymes.

In one embodiment, the nucleic acid encodes one or more enzymes in the mevalonate (MVA) pathway. In one embodiment, the one or more enzymes are
a) thiolase (EC 2.3.1.9),
b) HMG-CoA synthase (EC 2.3.3.10),
c) HMG-CoA reductase (EC 1.1.1.88),
d) Mevalonate kinase (EC 2.7.1.36),
e) phosphomevalonate kinase (EC 2.7.4.2),
f) selected from the group consisting of diphosphomevalonate decarboxylase (EC 4.1.1.33), and g) any variant thereof that is functionally equivalent.

  In certain embodiments, the nucleic acid encodes a thiolase (which may be an acetyl CoAc-acetyltransferase), HMG-CoA synthase, and HMG-CoA reductase.

In further embodiments, the nucleic acid encodes one or more enzymes in the mevalonate (MVA) pathway and one or more additional enzymes in the DXS pathway. In one embodiment, the one or more enzymes from the DXS pathway are
a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7),
b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267),
c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60),
d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148),
e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12),
f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1),
g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2), and h) selected from the group consisting of any variant thereof that is functionally equivalent Is done.

In a further embodiment, the nucleic acid encodes or expresses an exogenous nucleic acid or overexpressed endogenous that encodes or expresses one or more additional exogenous or endogenous enzymes that are expressed or overexpressed resulting in the production of a terpene compound or precursor thereof. Nucleic acids
a) Geranyltransferase Fps (EC: 2.5.1.10),
b) heptaprenyl diphosphate synthase (EC: 2.5.1.10),
c) Octaprenyl-diphosphate synthase (EC: 2.5.1.90),
d) isoprene synthase (EC 4.2.2.37),
e) Isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2),
f) an enzyme selected from the group consisting of farnesene synthase (EC 4.2.346 / EC 4.2.3.4) and any variant thereof that is functionally equivalent Code.

  In one embodiment, the nucleic acid encoding a thiolase (EC 2.3.1.9) has the sequence of SEQ ID NO: 40 or is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding a thiolase (EC 2.3.1.9) is an acetyl CoA c-acetyltransferase having the sequence of SEQ ID NO: 41 or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding HMG-CoA synthase (EC 2.3.3.10) has the sequence of SEQ ID NO: 42 or is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding HMG-CoA reductase (EC 1.1.1.88) has the sequence of SEQ ID NO: 43 or is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding mevalonate kinase (EC 2.7.1.36) has the sequence of SEQ ID NO: 51 or is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding phosphomevalonate kinase (EC 2.7.4.2) has the sequence of SEQ ID NO: 52 or is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding diphosphomevalonate decarboxylase (EC 4.1.1.13) has the sequence of SEQ ID NO: 53 or is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7) has the sequence of SEQ ID NO: 1 or is functionally equivalent thereof It is a mutant.

  In one embodiment, the nucleic acid encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267) has the sequence of SEQ ID NO: 3 or is functionally equivalent It is a mutant.

  In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60) has the sequence of SEQ ID NO: 5 or is functional It is a variant equivalent to.

  In one embodiment, the nucleic acid encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148) has the sequence of SEQ ID NO: 7 or is functionally equivalent It is a mutant.

  In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12) has the sequence of SEQ ID NO: 9 or is functional Is its equivalent.

  In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1) has the sequence of SEQ ID NO: 11 It is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2) has the sequence of SEQ ID NO: 13 or is functionally equivalent It is a mutant.

  In one embodiment, the nucleic acid encoding geranyltransferase Fps has the sequence of SEQ ID NO: 15, or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding heptaprenyl diphosphate synthase has the sequence of SEQ ID NO: 17, or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding octaprenyl-diphosphate synthase (EC: 2.5.1.90), wherein the octaprenyl-diphosphate synthase is a polyprenyl synthetase, is encoded by the sequence of SEQ ID NO: 19. Or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding isoprene synthase (ispS) has the sequence of SEQ ID NO: 21, or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding isopentenyl-diphosphate delta-isomerase (idi) has the sequence of SEQ ID NO: 54 or is a functionally equivalent variant.

  In one embodiment, the nucleic acid encoding farnesene synthase has the sequence of SEQ ID NO: 57 or is a functionally equivalent variant.

In one embodiment, the nucleic acid comprises the following enzymes a) isoprene synthase;
b) Isopentenyl-diphosphate Δ-isomerase (idi);
c) 1-deoxy-D-xylulose-5-phosphate synthase DXS;
Or encode functionally equivalent variants thereof.

In one embodiment, the nucleic acid comprises the following enzymes a) thiolase;
b) HMG-CoA synthase;
c) HMG-CoA reductase;
d) mevalonate kinase;
e) phosphomevalonate kinase;
f) diphosphomevalonate decarboxylase;
g) isopentenyl-diphosphate Δ-isomerase (idi); and h) isoprene synthase;
Or encode functionally equivalent variants thereof.

In one embodiment, the nucleic acid encodes the following enzymes: a) geranyltransferase Fps; and b) farnesene synthase or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid of the invention further comprises a promoter. In one embodiment, the promoter allows constitutive expression of the gene under its control. In certain embodiments, the Wood-Ljungdahl cluster promoter is used. In another specific embodiment, a phosphotransacetylase / acetate kinase operon promoter is used. In one particular embodiment, the promoter is C.I. It is derived from autoethanogenum.

  In a third aspect, the present invention provides a nucleic acid construct or vector comprising one or more nucleic acids of the second aspect.

  In one particular embodiment, the nucleic acid construct or vector is an expression construct or expression vector. In one particular embodiment, the expression construct or expression vector is a plasmid.

  In a fourth aspect, the present invention provides a host organism comprising any one or more of the nucleic acid of the second aspect or the vector or construct of the third aspect.

  In a fifth aspect, the present invention provides a composition comprising an expression construct or vector according to the third aspect of the invention and a methylation construct or vector.

  Preferably, the composition is capable of producing a recombinant microorganism according to the first aspect of the invention.

  In one particular embodiment, the expression construct / vector and / or methylation construct / vector is a plasmid.

  In a sixth aspect, the present invention provides one or more terpenes and / or precursors thereof by microbial fermentation comprising fermenting a substrate comprising CO using the recombinant microorganism of the first aspect of the present invention. Methods of producing the body and optionally one or more other products are provided.

In one embodiment, the method comprises:
(A) providing a substrate comprising CO to a bioreactor comprising a culture of one or more microorganisms of the first aspect of the invention; and (b) anaerobically fermenting the culture in the bioreactor. Producing at least one terpene and / or precursor thereof.

In one embodiment, the method comprises:
(A) supplementing the CO-containing gas produced as a result of the industrial process;
(B) anaerobically fermenting this CO-containing gas by culturing with one or more microorganisms of the first aspect of the invention to produce at least one terpene and / or precursor thereof.

  In certain embodiments of the method aspects, the microorganism is maintained in an aqueous culture medium.

  In a particular embodiment of this method aspect, the fermentation of the substrate takes place in a bioreactor.

  In one embodiment, the one or more terpenes and / or precursors thereof are mevalonic acid, IPP, dimethylallyl diphosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene. Selected from.

  Preferably, the substrate containing Co is a gaseous substrate containing CO. In one embodiment, the substrate comprises industrial waste gas. In certain embodiments, the gas is steel mill exhaust gas or synthesis gas.

  In one embodiment, the substrate is generally at least about 20 volume% to about 100 volume% CO, about 20 volume% to about 70 volume% CO, about 30 volume% to about 60 volume% CO, and about Includes major proportions of CO, such as 40-55 vol% CO. In certain embodiments, the substrate is about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or Contains about 60% CO by volume.

  In certain embodiments, the method further comprises recovering the terpene or precursor thereof and optionally one or more other products from the fermentation broth.

  In a seventh aspect, the present invention provides one or more terpenes and / or precursors thereof when produced by the method of the sixth aspect. In one embodiment, the one or more terpenes and / or precursors thereof are mevalonic acid, IPP, dimethylallyl diphosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene. Selected from the group consisting of

  In another aspect, the invention provides a method of producing a microorganism according to the first aspect of the invention, wherein the microorganism is fermented with a substrate comprising CO, wherein the microorganism is one or more terpenes and / or precursors thereof and optionally 1 Transforming a oxytrophic acetic acid-producing microorganism by introducing one or more nucleic acids so that one or more other products can be produced or increased, and the parental microorganism comprises CO A method is provided wherein one or more terpenes and / or precursors from fermentation cannot be produced or are produced at low levels.

  In one particular embodiment, the parental microorganism is transformed by introducing one or more exogenous nucleic acids adapted to express one or more enzymes in the mevalonate (MVA) pathway and optionally the DXS pathway. Is done. In another embodiment, the parental microorganism is adapted to overexpress one or more enzymes of the mevalonate (MVA) pathway and optionally the DXS pathway that are naturally present in the parental microorganism, using one or more nucleic acids. Transformed.

  In certain embodiments, the one or more enzymes are the enzymes previously described herein.

In one embodiment, an isolated and genetically modified oxytrophic acetic acid producing bacterium is provided, wherein the bacterium has an exogenous nucleic acid encoding an enzyme in the mevalonate pathway or DXS pathway or terpene biosynthesis pathway. And thereby the bacteria express the enzyme. The enzyme
a) thiolase (EC 2.3.1.9);
b) HMG-CoA synthase (EC 2.3.3.10);
c) HMG-CoA reductase (EC 1.1.1.88);
d) Mevalonate kinase (EC 2.7.1.36);
e) Phosphomevalonate kinase (EC 2.7.4.2);
f) Diphosphomevalonate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7);
g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267);
h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60);
i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148);
j) 2-C-methyl-D-erythritol 2; 4-cyclodiphosphate synthase IspF (EC: 4.6.1.12);
k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1);
l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2); geranyl transferase Fps (EC: 2.5.1.10);
m) heptaprenyl diphosphate synthase (EC: 2.5.1.10);
n) Octaprenyl-diphosphate synthase (EC: 2.5.1.90);
o) Isoprene synthase (EC 4.2.27);
p) isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2); and
q) Farnesene synthase (EC 4.2.3.46 / EC 4.2.3.
47)
Selected from the group consisting of

  In some embodiments, the bacterium does not express the enzyme in the absence of the nucleic acid. In some embodiments, the bacterium expresses the enzyme under anaerobic conditions.

One embodiment provides a plasmid that is capable of replicating in oxytrophic acetic acid producing bacteria. A plasmid comprises a nucleic acid encoding an enzyme in the mevalonate pathway or DXS pathway or terpene biosynthetic pathway, whereby the enzyme is expressed by the bacterium when the plasmid is present in the bacterium. The enzyme
a) thiolase (EC 2.3.1.9);
b) HMG-CoA synthase (EC 2.3.3.10);
c) HMG-CoA reductase (EC 1.1.1.88);
d) Mevalonate kinase (EC 2.7.1.36);
e) Phosphomevalonate kinase (EC 2.7.4.2);
f) Diphosphomevalonate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7);
g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267);
h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60);
i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148);
j) 2-C-methyl-D-erythritol 2; 4-cyclodiphosphate synthase IspF (EC: 4.6.1.12);
k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1);
l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2); geranyl transferase Fps (EC: 2.5.1.10);
m) heptaprenyl diphosphate synthase (EC: 2.5.1.10);
n) Octaprenyl-diphosphate synthase (EC: 2.5.1.90);
o) Isoprene synthase (EC 4.2.27);
p) Isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2); and q) Farnesene synthase (EC 4.2.3.4/EC 4.2.3.4)
Selected from the group consisting of

In another embodiment, a step of converting CO and / or CO ₂ to isoprene is provided. The process is such that the bacterium converts CO and / or CO ₂ to isoprene.
Includes passing the biological reactor CO-containing substrate and / or CO ₂ containing substrate of a gas containing a culture of carbon oxides nutritional acetate-producing bacteria in the culture medium, and recovering the isoprene from the bioreactor . Carbon-oxide-trophic acetic acid-producing bacteria are genetically modified to express isoprene synthase.

  In another embodiment, an isolated and genetically modified oxytrophic acetic acid producing bacterium comprising a nucleic acid encoding isoprene synthase is provided. Bacteria express isoprene synthase and bacteria can convert dimethylallyl diphosphate to isoprene. In one aspect, the isoprene synthase is a Populus tremuloides enzyme. In another aspect, the nucleic acid is an optimized codon. In yet another embodiment, the expression of isoprene synthase is under transcriptional control of the promoter of the pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum.

Another embodiment provides for converting CO and / or CO ₂ to isopentyl diphosphate (IPP). The process involves the inclusion of gaseous CO in a bioreactor containing a culture of oxytrophic acetic acid producing bacteria in a culture medium so that the bacteria convert CO and / or CO ₂ to isopentyl diphosphate (IPP). includes passing the substrate and / or CO ₂ containing substrate, and recovering the IPP from the bioreactor. Carbon oxide-trophic acetic acid producing bacteria have been genetically modified to express isoprene diphosphate Δisomerase.

  Yet another embodiment provides an isolated and genetically modified oxytrophic acetic acid producing bacterium comprising a nucleic acid encoding isopentyl diphosphate Δisomerase. Bacteria express isopentyl diphosphate Δ isomerase and bacteria can convert dimethylallyl diphosphate to isopentyl diphosphate. In some embodiments, the nucleic acid encodes Clostridium beijerinckii isopentyl diphosphate Δ isomerase. xxx missing linexx. In other embodiments, the nucleic acid is under transcriptional control of a promoter of a pyruvate: ferredoxin oxidoreductase gene derived downstream of a second nucleic acid encoding Clostridium autoethanogenum and isoprene synthase.

In yet another embodiment, a step of converting CO and CO ₂ to isopentyl diphosphate (IPP) and / or isoprene is provided. The process comprises a bioreactor comprising a culture of oxytrophic acetic acid producing bacteria in a culture medium such that the bacteria convert CO and / or CO ₂ to isopentyl diphosphate (IPP) and / or isoprene. to includes passing the CO-containing substrate and / or CO ₂ containing substrate gas, and recovering the IPP and / or isoprene from the bioreactor. Carbon-oxide-trophic acetic acid-producing bacteria have been genetically modified to increase the copy number of nucleic acid encoding the enzyme deoxyxylulose 5-phosphate synthase (DXS), with an increased copy number of 1 per genome. Over.

  Yet another embodiment is an isolated and genetically modified oxytrophic acetic acid production comprising more than one copy number per genome of nucleic acid encoding deoxyxylulose 5-phosphate synthase (DXS) enzyme Provide bacteria. In some embodiments, the isolated and genetically modified oxytrophic acetic acid producing bacterium may further comprise a nucleic acid encoding isoprene synthase. In other embodiments, the isolated and genetically modified oxytrophic acetic acid producing bacterium may further comprise a nucleic acid encoding isopentyl diphosphate Δ isomerase. In yet another aspect, the isolated and genetically modified oxytrophic acetic acid producing bacterium may further comprise a nucleic acid encoding isopentyl diphosphate Δ isomerase and a nucleic acid encoding isoprene synthase.

  Another embodiment provides an isolated and genetically modified oxytrophic acetic acid producing bacterium comprising a nucleic acid encoding phosphomevalonate kinase (PMK). The bacterium expresses the encoded enzyme and the enzyme is not from this bacterium. In some embodiments, the enzyme is a Staphylococcus aureus enzyme. In some embodiments, the enzyme has one or more C.I. It is expressed under the control of the autoethanogenum promoter. In some aspects, the bacterium further comprises a nucleic acid encoding a thiolase (thlA / vraB), a nucleic acid encoding an HMG-CoA synthase (HMGS), and a nucleic acid encoding an HMG-CoA reductase (HMGR). In some embodiments, the thiolase is a Clostridium acetobutyricum thiolase. In some embodiments, the bacterium further comprises a nucleic acid encoding diphosphomevalonate decarboxylase (PMD).

  In yet another embodiment, an isolated and genetically modified oxytrophic acetic acid producing bacterium comprising an exogenous nucleic acid encoding alpha-phenelsen synthase is provided. In some embodiments, the nucleic acid is C.I. Codon optimized for expression in autoethanogenum. In some embodiments, the alpha-phenelsen synthase is a Malus x domestica alpha-farnesene synthase. In some embodiments, the bacterium further comprises a nucleic acid segment encoding a geranyl transferase. In some embodiments, the geranyltransferase is an E. coli strain. E. coli geranyl transferase.

  Isolated and genetically modified carbon oxide-trophic acetic acid-producing bacteria suitable for any aspect or embodiment of the present invention are Clostridium autoethanogenum, Clostridium ljungdahldili, Clostridium carbosidoid, Clostridium carboxid, Clostridium carboxid , Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnesium, Butyracterium methanolicum, Acetobacterium woodody, Alkalibaculum b cchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, and Thermoanaerobacter may be selected from the group consisting of Kiuvi.

  The invention may also be broad to include, in part, the elements and features described or shown in the specification of the present application, which may be individual or collective, some of these, elements and It may be a combination of two or more of the features. Although certain integers are described herein, certain integers are described herein as having equivalents known in the art related to the present invention, and such equivalents are , Unless otherwise stated below, are incorporated herein.

  These and other aspects of the invention will be apparent from the following description, considered for all novel aspects and given by way of example only with reference to the accompanying drawings.

Is a pathway diagram of terpene production, gene targets described in this application are indicated by bold arrows Is the gene map of plasmid pMTL 85146-ispS. Figure 2 is a gene map of plasmid pMTL 85246-ispS-idi. Figure 2 is a gene map of plasmid pMTL 85246-ispS-idi-dxs. Fig. 4 shows the result of sequencing plasmid pMTL 85246-ispS-idi-dxs. Shows a comparison of the energetics of producing terpenes from CO via the DXS and mevalonate pathways. Indicates the mevalonate pathway. Is C.I. It is an image of the agarose gel electrophoresis which confirms presence of the isoprene expression of plasmid pMTL85246-ispS-idi in an autoethanogenum transformant. Lanes 1 and 20 show a 100 bp Plus DNA ladder. Lanes 3-6, 9-12, 15-18 show PCR using isolated plasmids from 4 different clones as templates, each in the following order: colE1, ermB, and idi. Lanes 2, 8, and 14 show PCR without template as a negative control, each in the following order: colE1, ermB, and idi. Lanes 7, 13, and 19 are E. coli as positive controls. The PCR using pMTL 85246 from E. coli is shown, each in the following order: colE1, ermB, and id. Shows the mevalonic acid expression plasmid MTL8215-Pptaack-thlA-HMGS-Patp-HMGR. Shows the isoprene expression plasmid pMTL 8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispS. Shows the farnesene expression plasmid pMTL8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS. Shows the gene map of plasmid pMTL 85246-ispS-idi-dxs. Has a plasmid pMTL 85146-ispS. The amplification chart of the experiment of gene expression using autoethanogenum is shown. Has a plasmid pMTL 85246-ispS-idi. The amplification chart of gene expression using autoethanogenum is shown. Has a plasmid pMTL 85246-ispS-idi-dxs. The amplification chart which experimented the gene expression using autoethanogenum is shown. Shows PCR confirming the presence of plasmid pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS. The size of the prediction band is 1584 bp. The DNA marker is a Fermentas 1 kb DNA ladder. Transformed with the plasmid pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS. It is the growth curve of autoethanogenmun. Are mevalonate kinase (MK SEQ ID NO: 51), phosphomevalonate kinase (PMK SEQ ID NO: 52), diphosphomevalonate decarboxylase (PMD SEQ ID NO: 53), isopentyl-diphosphate Δ-isomerase (idi SEQ ID NO: 54). ), RT-PRC data showing gene expression of geranyltransferase (ispA SEQ ID NO: 56) and farnesene synthase (Fs SEQ ID NO: 57). Is a GC-MS that detects and confirms the presence of farnesene in a culture supplemented with 1 mM mevalonic acid containing pMTL8314Prnf-MK-PMK-PMD-P of idi-ispA-FS. GC-MS black and white gram scanned peaks containing 93 ions of mass. Chromatograms 1 and 2 are transformed C.I. autoethanogenum, 3 is C.I. It is a β farnesene standard substrate that is introduced at the same time as the autoethanogenum sample. 4 shows E. coli with plasmid pMTL8314Prnf-MK-PMK-PMK-Pfor-idi-ispA-FS grown on M9 glucose, indicating α farnesene production. E. coli. 5 is an E.I. This is a standard substrate for β-farnesene that is introduced simultaneously with the E. coli sample. E. coli and C.I. The difference in retention time between autoethanogenum samples is due to minor changes to the analytical instrument. However, the retention time difference between the β-farnesene standard substrate and the produced α-farnesene is exactly the same in both cases, which together with the coincidence in the mass spectrum is C.I. The production of alpha farnesene of autoethanogenum is confirmed. Is the MS spectrum of the peaks labeled 1A and 2A in FIG. The MS spectrum is consistent with the NIST database spectrum (FIG. 21), confirming that the peak is alpha farnesene. Is the MS spectrum of alpha farnesene from the NIST mass spectral database.

  The following is a description of the present invention, including preferred embodiments thereof, using common language. The present invention is further elucidated from the disclosure under “Examples” herein below, the disclosure including experimental data supporting the present invention, specific examples of various aspects of the present invention, and the present invention. Means for carrying out the invention are provided.

  Surprisingly, the inventors have been able to modify oxytrophic acetic acid producing bacteria to produce terpenes and precursors including isoprene and farnesene by fermentation of a substrate including CO. This provides an alternative means for the production of these products that may have advantages over current methods for their production. Furthermore, we provide a means to use carbon monoxide from industrial processes that would otherwise be released to the atmosphere and pollute the environment.

  As used herein, “fermentation broth” refers to a culture medium that includes at least a nutrient medium and bacterial cells.

  As used herein, “shuttle microorganism” refers to a microorganism in which a methyltransferase enzyme is expressed and is different from the target microorganism.

  As used herein, “target microorganism” refers to a microorganism in which a gene contained on an expression construct / vector is expressed and is different from a shuttle microorganism.

  The term “main fermentation product” is intended to mean the one fermentation product produced at the highest concentration and / or yield.

  The terms “increased efficiency”, “increased efficiency” and the like, when used in connection with a fermentation process, include, but are not limited to, the growth rate of microorganisms catalyzing fermentation, increased product concentration. Of growth and / or product production rate, volume of desired product produced per volume of substrate consumed, production rate or level of desired product, and relative ratio of desired product compared to other by-products of fermentation Including at least one increase.

  “Substrate comprising carbon monoxide” and like terms should be understood to include, for example, any substrate for which carbon monoxide is available for growth and / or fermentation of one or more bacterial strains.

  “Gaseous substrate containing carbon monoxide” and similar terms and terms include any gas containing a level of carbon monoxide. In certain embodiments, the substrate comprises at least 20% to 100% CO, 20% to 70% CO, 30% to 60% CO, and 40% to 55% CO. . In certain embodiments, the substrate is about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or Contains about 60% CO by volume.

Although substrates need not include hydrogen, the presence of H ₂ is not deleterious to produce formed in accordance with the method of the present invention. In certain embodiments, the presence of hydrogen improves the overall efficiency of alcohol production. Xxmissing linexx For example, in certain embodiments, the substrate may comprise H ₂ : CO in a ratio of about 2: 1, or 1: 1, or 1: 2. In one embodiment, the substrate comprises about 30% or less H ₂ , about 20% or less H ₂ , about 15% or less H ₂ , or about 10% or less H ₂ . In other embodiments, the substrate of the flow, for example, less than 5%, or less than 4%, or less than 3%, or% less, or comprises less than the low concentration of H ₂ 1%, or hydrogen substantially Not included. The substrate also, for example, about 1 volume% to 80 volume%, or such as 1 volume% to about 30 volume% of CO _2, it may contain some CO _2. In one embodiment, the substrate comprises about 20% or less CO ₂ by volume. In certain embodiments, the substrate comprises about 15% or less CO ₂ , about 10% or less CO ₂ , about 5% or less CO ₂ , or substantially free of CO ₂ .

  As described below, embodiments of the present invention are described with respect to delivering and fermenting a “gaseous substrate comprising CO”. However, it should be understood that the gaseous substrate may be provided in alternative forms. For example, a gaseous substrate containing CO may be provided dissolved in a liquid. In essence, the liquid is saturated with a gas containing carbon monoxide, which is then added to the bioreactor. This can be achieved using standard methods. As an example, a fine bubble dispersion generator (Hensirisak et. Al. Scale-up of microbubble dispersion generator, aerobic fermentation can be used; Applied Biochemistry 3 Biomolecules and Biotechnolology. As a further example, a gaseous substrate comprising CO may be adsorbed on a solid support. Such alternative methods are included in the use of the term “substrate comprising CO” and similar terms.

  In certain embodiments of the invention, the CO-containing gaseous substrate is an industrial exhaust gas or an industrial waste gas. “Industrial waste gas or industrial exhaust gas” includes any gas containing CO produced by an industrial process, and also includes ferrous metal product manufacturing, non-ferrous product manufacturing, petroleum refining process, coal vaporization, biomass vaporization, power production, It should be broadly construed to include carbon black production and gases produced as a result of coke production. Further examples may be provided elsewhere herein.

  Unless otherwise required, the terms “fermentation”, “fermentation process”, or “fermentation reaction”, and the like, as used herein, are intended to include the growth and product biosynthesis stages of the process. Is done. As further described herein, in some embodiments, the bioreactor may include a first growth reactor and a second fermentation reactor. Thus, adding metals or compositions to the fermentation reaction should be understood to include addition to either or both of these reactors.

  The term “bioreactor” includes a fermentation apparatus consisting of one or more vessels and / or towers or pipe arrangements, a continuous stirred tank reactor (CSTR), an immobilized cell reactor (ICR), a trickle bed reactor. (TBR), bubble column reactors, gas lift fermenters, static mixers, or other equipment suitable for gas-liquid contact. In some embodiments, the bioreactor includes a first growth reactor and a second fermentation reactor. Thus, when referring to adding a substrate to a bioreactor or fermentation reaction, it is understood to include either or both of these reactors where appropriate.

  A “foreign nucleic acid” is a nucleic acid that originates outside the microorganism to be introduced. The exogenous nucleic acid is not limited, but the microorganism to be introduced (for example, the parent microorganism from which the recombinant microorganism is derived), the microorganism strain or type different from the organism to be introduced, or artificially or recombinantly created. It may be derived from any suitable source including microbial strains or species. In one embodiment, exogenous nucleic acids represent nucleic acid sequences that are naturally present in the introduced microorganism, and they are introduced to increase expression or overexpression of a particular gene (eg, a sequence (eg, a gene)). Or by introducing a strong or constitutive promoter to increase expression). In another embodiment, the exogenous nucleic acid represents a nucleic acid sequence that is not naturally present in the introduced microorganism and expresses a product that is not naturally present in the microorganism or increases the expression of a gene that is native to the microorganism ( For example, when introducing regulatory elements such as promoters). The exogenous nucleic acid may be adapted to integrate into the genome of the introduced microorganism or may be adapted to retain the state that is extrachromosomal.

  “Exogenous” may also be used to refer to a protein. This refers to a protein that is not present in the parental microorganism from which the recombinant microorganism is derived.

  The term “endogenous” as used herein with respect to recombinant microorganisms and nucleic acids or proteins refers to any nucleic acid or protein present in the parental microorganism from which the recombinant microorganism is derived.

  It is to be understood that the present invention may be practiced using nucleic acid sequences that differ from the sequences specifically exemplified herein, provided that they function substantially identically. For nucleic acid sequences that encode a protein or peptide, this means that the encoded protein or peptide has substantially the same function. For nucleic acid sequences representing promoter sequences, mutant sequences have the property of promoting expression of one or more genes. Such nucleic acids may be referred to herein as “functionally equivalent variants”. By way of example, functionally equivalent variants of a nucleic acid include allelic variants, gene fragments, mutations (deletions, insertions, nucleotide substitutions, etc.) and / or genes containing genetic polymorphisms, and the like. Homologous genes from other microorganisms may also be considered examples of functionally equivalent variants of the sequences specifically exemplified herein. These are Clostridium acetobutylicum, Clostridium beijerinckii, C.I. saccharobutyricum, and C.I. It includes homologous genes of species such as Saccharoperbutylacetonicum, details of which are published and available on websites such as GeneBank or NCBI. The term “functionally equivalent variants” should also be construed to include nucleic acids whose sequences have changed as a result of codon optimization for a particular organism. “Functionally equivalent variants” of the nucleic acids described herein are preferably at least about 70%, preferably 80%, more preferably 85%, preferably about 90%, of the identified nucleic acid sequence, Preferably it has about 95% or more nucleic acid sequence identity.

  It should also be understood that the present invention may be practiced using polypeptides whose sequences differ from the amino acid sequences specifically exemplified herein. These variants may be referred to herein as “functionally equivalent variants”. A functionally equivalent variant of a protein or peptide is at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 80%, preferably with the identified protein or peptide. Includes proteins or peptides that share 90%, preferably 85%, preferably 90%, preferably 95% or more amino acid identity and have substantially the same function as the protein or peptide of interest. Such variants include those that are within the scope of a protein or peptide fragment, the fragment includes a truncated form of the polypeptide, and the deletion is 1-5, -10, -15, -20, -25 It may be an amino acid and may extend from 1 to 25 residues at either end of the polypeptide, and the deletion may be any length within the region; May be. Functionally equivalent variants of a particular polypeptide herein should also be construed to include polypeptides expressed by homologous genes of other bacterial species, for example as exemplified in the previous paragraph. It is.

  As used herein, “substantially identical function” is intended to mean that a variant of a nucleic acid or polypeptide can perform the function of the original nucleic acid or polypeptide. For example, a variant of the enzyme of the invention can catalyze the same reaction as that enzyme. However, this does not mean that the variant has the same level of activity as the original polypeptide or nucleic acid.

  Whether a functionally equivalent variant has substantially the same function as the original nucleic acid or polypeptide can be assessed using any known method. However, by way of example, the method described by Silver et al. For isoprene synthase (1991, Plant Physiol. 97: 1588-1591) or the method described by Zhao et al. (2011, Appl Microbiol Biotechnol, 90: 1915- 1922), The method by Green et al. For farnesene synthase (2007, Phytochemistry; 68: 176-188), the method by Kuzuyama for 1-deoxy-D-xylulose 5-phosphate synthase Dxs (2000, J. Bacteriol. 182, 891-897). ), The method by Berndt and Schlegel for thiolase (1975, Arch. Microbiol). 103, 21-30) or the method by Stim-Herndon et al. (1995, Gene 154: 81-85), the method by Cabano et al. For HMG-CoA synthase (1997, Insect Biochem. Mol. Biol. Metab. 27: 499-). 505), the method by Ma et al. For HMG-CoA reductase and mevalonate kinase enzyme (2011, Metab. Engin., 13: 588-597), the method by Herdendorf and Miziokko for phosphomevalonate kinase (2007, Biochemistry 17:46: -8), the method by Krepkiy et al. For diphosphomevalonate decarboxylase (2004, Protein Sc . 13: 1875-1881), and the like. It is also possible to identify genes in the DXS and mevalonate pathway using inhibitors such as fosmidomycin or mevinolin, as described by Truko et al. (2005, Microbiology 74: 153-158). .

  As used herein, terms and phrases such as “overexpression”, “overexpression”, and the like refer to one or more expression levels compared to the expression level of a parental microbial protein (including nucleic acids) under the same conditions. It should be construed broadly to include any increase in protein expression (including expression of one or more nucleic acids encoding the same protein). It should not mean that the protein (or nucleic acid) is expressed at any particular level.

  A “parental microorganism” is a microorganism used to make the recombinant microorganism of the present invention. A parental microorganism is one that is naturally occurring (ie, a wild-type microorganism) or one that has been previously modified but does not express or overexpress one or more enzymes that are the subject of the present invention. Accordingly, the recombinant microorganisms of the present invention may be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism.

  Terms such as nucleic acid “construct” or “vector” should be broadly construed to include any nucleic acid (including DNA and RNA) suitable for use as a vehicle to transfer genetic substrates into cells. The term should be construed to include plasmids, viruses (including bacteriophages), cosmids and artificial chromosomes. A construct or vector may contain one or more regulatory elements, origins of replication, multiple cloning sites and / or selectable markers. In one embodiment, the construct or vector is adapted to allow expression of one or more genes encoded by the construct or vector. Nucleic acid constructs or vectors include naked nucleic acids as well as nucleic acids (eg, liposome-bound nucleic acids, organisms containing nucleic acids) formulated with one or more agents that facilitate delivery to cells.

  A “terpene” as referred to herein includes any compound made from linked C5 isoprene units, including simple and complex terpenes and oxygen-containing terpene compounds such as alcohols, aldehydes and ketones. Should be interpreted widely. Simple terpenes are found in essential oils and resins of plants such as conifers. More complex terpenes include terpenoids and vitamin A, carotenoid pigments (such as lycopene), squalene, and gum. Examples of monoterpenes include, but are not limited to isoprene, pinene, nerol, citral, camphor, menthol, limonene. Examples of sesquiterpenes include, but are not limited to, nerolidol and farnesol. Examples of diterpenes include, but are not limited to, phytol and vitamin A1. Squalene is an example of a triterpene, and carotene (provitamin A1) is a tetraterpene.

  A “terpene precursor” is a compound or intermediate material produced during a reaction to form a terpene initiated from acetyl CoA and optionally pyruvate. The term refers to precursor compounds or intermediate substrates found in the mevalonic acid (MVA) pathway and optionally the DXS pathway and precursors downstream of longer chain terpenes such as FPP and GPP. Specific embodiments include, but are not limited to, mevalonic acid, IPP, dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP).

  The “DXS pathway” is an enzymatic pathway from pyruvate and D-glyceraldehyde-3-phosphate to DMAPP or IPP. It is also known as the deoxyxylulose 5-phosphate (DXP / DXPS / DOXP or DXS) / methylerythritol phosphate (MEP) pathway.

  The “mevalonic acid (MVA) pathway” is an enzymatic pathway from acetyl Coa to IPP.

Microorganisms Deoxyxylulose 5-phosphate (DXP / DXPS) starting from two terpene production pathways, two important intermediate substrates in glycolysis, pyruvate and D-glyceraldehyde-3-phosphate (G3P) / DOXP or DXS) / methylerythritol phosphate (MEP) pathway (Hunter et al., 2007, J. Biol. Chem. 282: 21573-77), and the mevalonic acid (MVA) pathway (Miziorko, starting from acetyl-CoA) 2011, Arch Biochem Biophys, 505: 131-143). Many different classes of microorganisms have been studied for the presence of either of these pathways (Lange et al., 2000, PNAS, 97: 13172-77; Trutko et al., 2005, Microbiology, 74: 153-158. Julsing et al., 2007, Appl Microbiol Biotechnol, 75: 1377-84) has not been studied in oxytrophic acetic acid production. For example, the DXS path is E. has been found to be present in E. coli, Bacillus, or Mycobacterium, while the mevalonic acid pathway is present in the yeast Saccharomyces, Cloroflexus, or Myxococcus.

Carbon oxide nutrient acetic acid producing C.I. autoethanogenum, C.I. The ljungdahlii genome was analyzed by the inventors for the presence of either of the two pathways. All genes of the DXS pathway are C.I. autoethanogenum and C.I. identified in ljungdahlii (Table 1), while the mevalonate pathway was absent. Furthermore, C.I. autoethanogenum or C.I. Production of oxytrophic acetic acid such as ljungdahlii is not known to produce any terpene as a metabolic end product.

Genes for downstream terpene synthesis from isoprene units were also identified in both organisms (Table 2).

  Terpenes are energy-concentrating compounds, and their synthesis requires cells to spend energy in the form of nucleoside triphosphates such as ATP. The use of sugar as a substrate requires sufficient energy supplied from glycolysis to obtain several molecules of ATP. Production of terpenes and / or their precursors via the DXS pathway using sugar as a substrate is a relatively simple method due to the availability of pyruvate and D-glyceraldehyde-3-phosphate (G3P) G3P is derived from C5 pentose and C6 hexose sugars. These C5 and C6 molecules are therefore relatively easily converted into C5 isoprene units from which terpenes are constructed.

  In anaerobic acetic acid production using C1 substrates such as CO or CO2, it is more difficult to synthesize long molecules such as hemiterpenoids from C1 units. This is especially true for longer chain terpenes such as C10 monoterpenes, C15 sesquiterpenes, or C40 tetraterpenes. The product with the most carbon atoms reported so far in acetic acid producing bacteria (both natural and recombinant microorganisms) is the C4 compound butanol (Kopke et al., 2011, Curr. Opin. Biotechnol. 22: 320-). 325; Schiel-Bengelsdorf and Durre, 2012, FEBS Letters: 10.1016 / j.febslet.2012.4.043; Kopke et al., 2011, Proc.Nat.Sci. 92; U.S. Patent No. 2011/0236941) and 2,3-butanediol (Koppe et al., 2011, Appl. Environ. Microbiol. 77: 5467-75). It is. The inventors have shown that it is surprisingly possible to anaerobically produce these longer chain terpenes using a C1 feedstock CO via an acetyl CoA intermediate.

The energetics of the Wood-Ljungdahl pathway in anaerobic acetic acid producers is just becoming clear, but unlike anaerobic growth conditions or glycolysis in sugar-fermenting organisms, the Wood-Ljungdahl pathway is ATP in the Wood-Ljungdahl pathway by phosphorylation at the substrate level. In fact, the activation of CO ₂ to formic acid requires essentially one molecule of ATP and requires a membrane gradient. The inventors point out that it is important that the pathway for product formation is energy efficient. We found that in acetic acid producing bacteria, the substrate CO or CO ₂ is directly transferred to acetyl CoA, which requires only 6 reactions, especially when compared to sugar-based systems, and terpenes and / or their Note that it represents the most direct route to the precursor (Figure 1). Although less ATP is available in anaerobic acetic acid producing bacteria, we have found that this more direct pathway sustains higher metabolic flow (due to the higher chemical driving force of the intermediate reaction). I think. Higher metabolic streams are important because some intermediates in the terpene biosynthetic pathway, such as the key intermediates mevalonic acid and FPP, are toxic to many bacteria if not efficiently turned over.
Despite the availability of higher ATP, this intermediate toxicity problem can be an obstacle in terpene production from sugars.

  Comparing the energetics of terpene precursors IPP and DMAPP production from CO via the mevalonate pathway with the DXS pathway, the mevalonate pathway requires less nucleoside triphosphates as ATP, less equivalent reduction, and We point out that only 6 reaction steps from acetyl-CoA are required and are also more direct compared to the DXS pathway. This provides an advantage to the rate of reaction and metabolic flow and increases overall energy efficiency. Furthermore, the smaller number of enzymes required simplifies the recombination method required to produce recombinant microorganisms.

  Although acetic acid producing bacteria with the mevalonate pathway have not been identified, we have established the mevalonate pathway and optionally the DXS pathway as Clostridium autoethanogenum or C. cerevisiae. It has been shown that it is possible to efficiently produce terpenes and / or precursors thereof from CO, which is a C1 carbon substrate, by introducing into carbon oxide-trophic acetic acid-producing bacteria such as ljungdahlii. This is expected to be applicable to all carbon oxide-trophic acetic acid producing microorganisms.

  Furthermore, the production of terpenes and / or their precursors has never been shown to be possible using recombinant microorganisms under anaerobic conditions. The anaerobic production of isoprene has the advantage of providing a safer operating environment. Because isoprene is highly flammable in the presence of oxygen, it has a lower flammability limit (LFL) of 1.5-2.0% and an upper flammability limit of 2.0-12% at room temperature and atmospheric pressure. This is because (UFL) is included. Because combustion does not occur without oxygen, we believe that an anaerobic combustion process is desirable because it is safer over all product concentrations, gas compositions, temperatures and pressure ranges.

  As previously described herein, the present invention provides a recombinant microorganism capable of producing one or more terpenes and / or precursors thereof, and optionally one or more other products, by fermentation of a substrate comprising CO. provide.

In a further embodiment, the microorganism is
Expresses one or more exogenous enzymes from the mevalonate (MVA) pathway and / or overexpresses one or more endogenous enzymes from the mevalonate (MVA) pathway; and a) 1 from the DXS pathway It is adapted to express one or more exogenous enzymes and / or to overexpress one or more endogenous enzymes from the DXS pathway.

  In one embodiment, the parental microorganism from which the recombinant microorganism is derived can ferment a substrate containing CO to produce acetyl CoA, but cannot convert acetyl CoA to mevalonic acid or isopentyl pyrophosphate (IPP), and the recombinant microorganism Are adapted to express one or more enzymes involved in the mevalonate pathway.

  The microorganism can, for example, increase the expression of a natural gene in the microorganism (eg, by introducing a stronger or constitutive promoter to drive gene expression), encode the enzyme and Increasing the copy number of a gene encoding a particular enzyme by introducing an exogenous nucleic acid adapted to express, and an exogenous adapted to encode and express an enzyme that does not naturally occur in the parental microorganism One or more enzymes may be adapted to be expressed or overexpressed by any number of recombinant methods, including introducing a sex nucleic acid.

In one embodiment, the one or more enzymes are from the mevalonate (MVA) pathway and a) a thiolase (EC 2.3.1.9),
b) HMG-CoA synthase (EC 2.3.3.10),
c) HMG-CoA reductase (EC 1.1.1.88),
d) Mevalonate kinase (EC 2.7.1.36),
e) phosphomevalonate kinase (EC 2.7.4.2),
f) selected from the group consisting of diphosphomevalonate decarboxylase (EC 4.1.1.33), and g) any variant thereof that is functionally equivalent.

In a further embodiment, any one or more enzymes are from the DXS pathway, and a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7),
b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267),
c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60),
d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148),
e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12),
f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1),
g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2), and h) selected from the group consisting of any variant thereof that is functionally equivalent Is done.

In further embodiments, one or more exogenous or endogenous additional enzymes are expressed or overexpressed resulting in the production of terpene compounds and / or precursors thereof, expressed exogenous enzymes, or overexpressed. Endogenous enzymes
a) Geranyltransferase Fps (EC: 2.5.1.10),
b) heptaprenyl diphosphate synthase (EC: 2.5.1.10),
c) Octaprenyl-diphosphate synthase (EC: 2.5.1.90),
d) isoprene synthase (EC 4.2.2.37),
e) Isopentenyl-diphosphate Δ-isomerase (EC EC 5.3.3.2),
f) Farnesene synthase (EC 4.2.3.4/EC 4.2.3.4), and g) any variant thereof that is functionally equivalent.
Selected from the group consisting of

  For illustration purposes only, the sequence information for each enzyme is listed in the drawings herein.

  The enzymes used in the microorganisms of the present invention may be from any suitable source, including different genera and species of bacteria, or other organisms. However, in one embodiment, the enzyme is from Staphylococcus aureus.

  In one embodiment, the enzyme isoprene synthase (ispS) is derived from Poplar tremuloides. In a further embodiment, it has the nucleic acid sequence exemplified in SEQ ID NO: 21 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme deoxyxylulose 5-phosphate synthase is C.I. Those derived from autoethanogenum, having the amino acid sequence exemplified by SEQ ID NO: 1 and / or exemplified by SEQ ID NO: 2, or functionally equivalent variants thereof.

  In one embodiment, the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 3 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 5 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 7 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 9, or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 11 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 13 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme mevalonate kinase (MK) is Staphylococcus aureus subsp. Aureus Mu50-derived and encoded by the nucleic acid sequence exemplified in SEQ ID NO: 51, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme phosphomevalonate kinase (PMK) is staphylococcus aureus subsp. It is derived from aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 52 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme diphosphomevalonate decarboxylase (PMD) is staphylococcus aureus subsp. It is derived from aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 53 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme isopentenyl-diphosphate Δ-isomerase (idi) is derived from Clostridium beijerinckii and is encoded by a nucleic acid sequence exemplified in SEQ ID NO: 54 below, or a functionally equivalent variant thereof Is the body.

  In one embodiment, the enzyme thiolase (thIA) is derived from Clostridium acetobutyricum ATCC 824 and is encoded by a nucleic acid sequence exemplified in SEQ ID NO: 40 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme is a thiolase enzyme and Staphylococcus aureus subsp. Aureus Mu50-derived acetyl-CoA c-acetyltransferase (vraB), which is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 41 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is obtained from Staphylococcus aureus subsp. It is derived from aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 42 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme hydroxymethylglutaryl-CoA reductase (HMGR) is staphylococcus aureus subsp. It is derived from aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 43 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme geranyl transferase (ispA) is Escherichia coli str. K-12 substr. It is derived from MG1655 and is encoded by a nucleic acid sequence exemplified by SEQ ID NO: 56 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme heptaprenyl diphosphate synthase is C.I. It is derived from autoethanogenum and is encoded by a nucleic acid sequence exemplified in SEQ ID NO: 17 below, or a functionally equivalent variant thereof.

  In one embodiment, the enzyme polyprenyl synthetase is C.I. It is derived from autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 19 or is a functionally equivalent variant thereof.

  In one embodiment, the enzyme α-farnesene synthase (FS) is derived from Malus x domestica and is encoded by a nucleic acid sequence exemplified in SEQ ID NO: 57 below, or a functionally equivalent variant thereof. .

  Enzymes and functional variants used in microorganisms may be identified by assays known to those skilled in the art. In certain embodiments, the enzyme isoprene synthase is identified by the method summarized by Silver et al. (1991, Plant Physiol. 97: 1588-1591) or Zhao et al. (2011, Appl Microbiol Biotechnol, 90: 1915- 1922). Also good. In a further specific embodiment, farnesene synthase may be identified by the method summarized by Green et al. (2007, Phytochemistry; 68: 176-188). In a further specific embodiment, the enzyme from the mevalonate pathway is a method according to Cabano et al. (1997, Insect Biochem. Mol. Biol. 27: 499-505) for HMG-CoA synthase, HMG-CoA reductase and mevalonate kinase. Ma et al. (2011, Metab. Engin., 13: 588-597), Herdendorf and Miziorko method with phosphomevalonate kinase (2007, Biochemistry, 46: 11780-8), and Krepki et al. With diphosphomevalonate decarboxylase. Method (2004, Protein Sci. 13: 1875-1881), Ma et al. 2011, Me tab. Engin. 13: 588-597. The 1-deoxy-D-xylulose 5-phosphate synthase of the DXS pathway can be assayed using the method summarized by Kuzuyama et al. (2000, J. Bacteriol. 182, 891-897). Also, identifying inhibitors of the DXS and mevalonate pathways using inhibitors such as fosmidomycin or mevinolin as described by Truko et al. (2005, Microbiology 74: 153-158). Is also possible.

  In one embodiment, the microorganism comprises one or more exogenous nucleic acids adapted to increase the expression of one or more endogenous nucleic acids, and the one or more endogenous nucleic acids are as previously described herein. Encodes one or more of the enzymes In one embodiment, the one or more exogenous nucleic acids adapted to increase expression is a regulatory element. In one embodiment, the regulatory element is a promoter. In one embodiment, the promoter is a constitutive promoter that is preferably highly active under suitable fermentation conditions. Inducible promoters can also be used. In a preferred embodiment, the promoter is selected from the group comprising the Wood-Ljungdahl gene cluster or the phosphotransacetylase / acetate kinase operon promoter. One skilled in the art will appreciate that other promoters capable of directing expression, preferably high levels of expression under suitable fermentation conditions, are useful as an alternative to exemplary embodiments.

  In one embodiment, the microorganism comprises one or more exogenous nucleic acids that are adapted to encode and express one or more enzymes previously described herein. In one embodiment, the microorganism is adapted to encode and express at least two enzymes and includes one or more exogenous nucleic acids. In other embodiments, the microorganism encodes and expresses at least 3, at least 4, at least 5, at least 5, at least 6, at least 7, at least 8, at least 9 or more enzymes. It is adapted and contains one or more exogenous nucleic acids.

  In one particular embodiment, the microorganism comprises one or more exogenous nucleic acids encoding an enzyme of the invention or a functionally equivalent variant thereof.

  The microorganism may contain one or more exogenous nucleic acids. If it is desirable to transform a parental microorganism with more than one genetic component (such as a gene or a regulatory element (eg, a promoter)), they may be included in one or more exogenous nucleic acids.

  In one embodiment, the one or more exogenous nucleic acids are nucleic acid constructs or vectors, and in one particular embodiment, a plasmid that encodes one or more of the enzymes described herein in any combination. is there.

  The exogenous nucleic acid may be retained outside the chromosome during the transformation of the parent microorganism, or may be integrated into the genome of the parent microorganism. Thus, the exogenous nucleic acid supports the expression and replication of additional nucleotide sequences adapted to support integration (eg, regions that allow homologous recombination and targeted integration into the host genome) or extrachromosomal constructs. Additional nucleotide sequences adapted to do so (eg, origin of replication, promoter and other regulatory elements or sequences) may be included.

  In one embodiment, the exogenous nucleic acid encoding one or more enzymes previously described herein further comprises a promoter adapted to promote expression of the one or more enzymes encoded by the exogenous nucleic acid. In one embodiment, the promoter is a constitutive promoter that is preferably highly active under suitable fermentation conditions. Inducible promoters can also be used. In a preferred embodiment, the promoter is selected from the group comprising the Wood-Ljungdahl gene cluster and the phosphotransacetylase / acetate kinase promoter. One skilled in the art will appreciate that other promoters capable of directing expression, preferably high levels of expression under suitable fermentation conditions, are useful as an alternative to the illustrated embodiments.

  In one embodiment, the exogenous nucleic acid is an expression plasmid.

  In one particular embodiment, the parental microorganism is selected from the group of oxytrophic acetic acid producing bacteria. In certain embodiments, the microorganism, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium lim Osum, Moorella thermoacetica, Moorella thermoautotropica, Sporumusa ovata, Sporousa sylvaetica, Sporomusa sphaeroides, Oxobacter pfennigi, and Toxobacter pfennigi.

In one particular embodiment, the parental microorganism is C.I. autoethanogenum, C.I. ljungdahlii, C.I. Selected from clusters of ethanol-producing, acetic acid-producing Clostridium, including ragsdalei and related isolates. These include, but are not limited to, the established cell line C.I. autoethanogenum JAI-1T DSM10061) [Abrini J, Naveau H, Nyns EJ: Clostridium autoethanogenum, sp. nov. , Anaerobic battery that products ethanol carbon monoxide. Arch Microbiol 1994, 4: 345-351], C.I. autoethanogenum LBS 1560 (DSM 19630) [Simpson SD, Forster RL, Tran PT, Rowe MJ, Warner IL: Novell bacteria and methods theof. International patent 2009, WO / 2009/064200], C.I. autoethanogenum LBS 1561 (DSM23693), C.I. ljungdahlii PETC ^T (DSM 13528 = ATCC 55383) [Tanner RS, Miller LM, Yang D: Clostridium ljungdahlii sp. nov. , An Acetogenic Species in Clostrial rRNA Homology Group I. Int J Systact Bacteriol 1993, 43: 232-236], C.I. ljungdahlii ERI-2 (ATCC 55380) [Gaddy JL: Clostridium stain what products acetic acid from waste gases. US patent 1997, 5,593,886], C.I. ljungdahlii C-01 (ATCC 55988) [Gaddy JL, Clausen EC, Ko C-W: Microbial process for the preparation of the world as well as the solvent. US patent, 2002, 6, 368, 819], C.I. ljungdahlii O-52 (ATCC 55989) [Gaddy JL, Clausen EC, Ko C-W: Microbial process for the preparation of the world as well as the solvent. US patent, 2002, 6, 368, 819], C.I. ragsdalei P11 ^T (ATCC BAA-622) [Hunnke RL, Lewis RS, Tanner RS: Isolation and Characteristic of novel Clostritional Species. International patent 2008, WO 2008/028055], “C. coskatati” [Zahn et al-Novel ethanolic technology estro um Jr. U.S. 201 and U.S. Patent Application. Res.4: 1-12) or related isolates such as C.I. ljungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina Ut. These strains form sub-clusters within Clostridium rRNA cluster 1, and their 16S rRNA genes are similar to more than 99% with similar low GC content around 30%. However, DNA-DNA reassociation and DNA fingerprinting experiments indicated that these strains belonged to different species [Hunnke RL, Lewis RS, Tanner RS: Isolation and Characteristic of novel Crossspecials. International patent 2008, WO 2008/028055].

  All species of this cluster have similar morphology and size (logarithmic growth phase cells are 0.5-0.7 × 3-5 μm) mesophilic (optimal growth temperature 30-37 ° C.) and strictly Anaerobic [Tanner RS, Miller LM, Yang D: Clostridium ljungdahlii sp. nov. , An Acetogenic Species in Clostrial rRNA Homology Group I. Int J Systact Bacteriol 1993, 43: 232-236; Abrin J, Naveau H, Nyns EJ: Clostridium autoethanogenum, sp. nov. , Anaerobic battery that products ethanol carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke RL, Lewis RS, Tanner RS: Isolation and Characteristic of novel Crossspecials. International patent 2008, WO 2008/028055]. Furthermore, they have the same pH range (pH 4-7.5, optimum initial pH: 5.5-6), similar growth profile and similar metabolic profile with ethanol and acetic acid as main fermentation end products, CO-containing gas Share similar major systematic properties such as strong autotrophic growth at low pressure and small amounts of 2,3-butanediol and lactic acid formed under certain conditions [Tanner RS, Miller LM, Yang D: Clostridium ljungdahlii sp. nov. , An Acetogenic Species in Clostrial rRNA Homology Group I. Int J Systact Bacteriol 1993, 43: 232-236; Abrin J, Naveau H, Nyns EJ: Clostridium autoethanogenum, sp. nov. , Anaerobic battery that products ethanol carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke RL, Lewis RS, Tanner RS: Isolation and Characteristic of novel Crossspecials. International patent 2008, WO 2008/028055]. Indole production was also observed in all three species. However, this species is in use with substrates of various sugars (eg rhamnose, arabinose), acids (eg gluconic acid, citric acid), amino acids (eg arginine, histidine), or other substrates (eg betaine, butanol). There is a difference. In addition, some species have been found to be autotrophic for certain vitamins (eg thiamine, biotin), while others are not.

  In one embodiment, the parent carbon oxides nutritive acid-producing microorganism, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moo ella thermautotrophica, is selected from the group consisting of Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

  In one particular embodiment of the first or second aspect, the parent microorganism, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, carbon oxides containing Clostridium magnum Selected from the group of nutritional Clostridium.

In one embodiment, the microorganism is C.I. autoethanogenum, C.I. Selected from a cluster of oxytrophic Clostridium, including ljungdahlii, and “C. ragsdalei” and related isolates. These include but are not limited to bacterial strains C.I. autoethanogenum JAI-1 ^T (DSM10061) (Abrini, Naveau, & Nyns, 1994), C.I. autoethanogenum LBS1560 (DSM19630) (WO / 2009/064200), C.I. autoethanogenum LBS 1561 (DSM23693), C.I. ljungdahlii PETCT (DSM 13528 = ATCC 55383) (Tanner, Miller, & Yang, 1993), C.I. ljungdahlii ERI-2 (ATCC 55380) (US Pat. No. 5,593,886), C.I. ljungdahlii C-01 (ATCC 55988) (US Pat. No. 6,368,819), C.I. ljungdahlii O-52 (ATCC 55989) (US Pat. No. 6,368,819), or “C. ragsdalei P11 ^T ” (ATCC BAA-622) (WO 2008/028055), and “C. coskatii” (US patent) No. 2011/0229947), “Clostridium sp. MT351” (Michael Tyurin & Kiriukin, 2012) and related isolates such as C.I. ljungdahlii OTA-1 (including the Tirado-Acevedo O. Production of Bioethanol from Synthesis Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina U.S.

  Despite being of different types as determined by DNA-DNA reassociation and DNA fingerprinting experiments, these strains have at least 99% identity at the 16S rRNA gene level and have a Clostridial rRNA cluster I Form sub-clusters within. (Collins et al., 1994) (WO 2008/028055, US Pat. No. 2011/0229947)

  Strains of this cluster have both similar genotypes and phenotypes, are defined by common features, and share the same mode of energy conservation and fermentation metabolism. This cluster strain lacks cytochrome and conserves energy via the Rnf complex.

  Strains of this cluster have a genome size of around 4.2 MBp (Kopke et al., 2010) and a GC composition of around 32 mol% (Abrini et al., 1994; Kopke et al., 2010; Tanner et al., 1993) (WO 2008/028055; US patent 2011/0229947), and an essential gene operon encoding the Wood-Ljungdahl pathway enzyme is conserved (carbon monoxide dehydrogenase, formyltetrahydrofolate) Synthetase, methylene-tetrahydrofolate dehydrogenase, formyl-tetrahydrofolate cyclohydrolase, methylene-tetrahydrofolate reductase, and carbon monoxide dehydrogenase / acetyl-Co A synthase), hydrogenase, formate dehydrogenase, Rnf complex (rnfCDGEAB), pyruvate: ferredoxin oxidoreductase, aldehyde: ferredoxin oxidoreductase (Kopke et al., 2010, 2011). The organization and number of the Wood-Ljungdahl pathway genes are found to be the same in all species, despite involvement in gas uptake and differences in nucleic acid and amino acid sequences (Kopke et al. , 2011).

  These strains have similar morphology and size (logarithmic growth phase cells are 0.5-0.7 × 3-5 μm), are mesophilic (optimal growth temperature 30-37 ° C.), And strictly anaerobic (Abrini et al., 1994; Tanner et al., 1993) (WO 2008/028055). Furthermore, they all have the same pH range (pH 4-7.5 optimum initial pH 5.5-6), strong autotrophic growth with CO-containing gases at similar growth ratios, as well as small amounts formed under certain conditions Share the same main phylogenetic properties, such as metabolic profile with ethanol and acetic acid as the end products of the main fermentation, with 2,3-butanediol and lactic acid (Abrini et al., 1994; Kopke et al , 2011; Tanner et al., 1993). However, these species are in use with substrates of various sugars (eg rhamnose, arabinose), acids (eg gluconic acid, citric acid), amino acids (eg arginine, histidine), or other substrates (eg betaine, butanol). There is a difference. Some species have been found to be auxotrophic for certain vitamins (eg thiamine, biotin), while others are not. Reduction of carboxylic acids to the corresponding alcohols has been shown in a variety of these organisms (Perez, Richter, Loftus, & Agent, 2012).

  The described features include C.I. autoethanogenum or C.I. It is not specific to one organism such as ljungdahlii, but rather is a general feature of carbon oxide-trophic ethanol-synthetic clostridial. Thus, the present invention may be expected to work across these strains, although there may be differences in performance.

  The recombinant carbon oxytrophic acetic acid producing microorganism of the present invention can be any number of techniques known to those of skill in the art for producing recombinant microorganisms from a parent carbon oxytrophic acetic acid producing microorganism and one or more exogenous nucleic acids. May be used to prepare. By way of example only, transformation (including transduction or transfection) has been achieved by electroporation, electrofusion, sonication, polyethylene glycol mediated transformation, conjugation, or chemical and natural competence. Also good. Suitable transformation techniques are described, for example, in Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold 9 Spring Spring, 198.

  Electroporation is a C.I. ljungdahlii (Kopke et al., 2010; Leang, Ueki, Nevin, & Lovley, 2012) (PCT / NZ2011 / 000203; WO2012 / 053905), C.I. autoethanogenum (PCT / NZ2011 / 000203; WO2012 / 053905), Acetobacterium woodi (Stratz, Sauer, Kuhn, & Durre, 1994) or Moorella thermoacetica (alkaline acid production) And C.I. acetobutylicum (Mermelstein, Welker, Bennett, & Papoutakis, 1992), C.I. cellulolyticum (Jennert, Tardif, Young, & Young, 2000) or C.I. It is a standard method used in many Clostridium bacteria, such as thermocellum (MV Tyurin, Desai, & Lynd, 2004).

  Electrofusion is performed by acetic acid producing bacteria Clostridium sp. MT351 has been described (Tyurin and Kiriukin, 2012).

  The introduction of prophage is C. scatologenes (Prasanna Tamarapu Parthasarathy, 2010, Development of a Genetic Modification System in Clostridium scatologenes ATCC 25775 for Generation of Mutants, Masters Project Western Kentucky University) have been described for similarly oxidized carbon nutritional acetate-producing bacteria in the case of.

  Conjugation was performed by acetic acid producing bacteria Clostridium difficile (Herbert, O'Keeffe, Purdy, Elmore, & Minton, 2003) and C.I. It has been described as the method of choice for many other clostridiums, including acetobulycum (Williams, Young, & Young, 1990).

  In one embodiment, the parent strain uses CO as the sole carbon and energy source.

  In one embodiment, the parental microorganism is Clostridium autoethanogenum or Clostridium ljungdahlii. In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693. In another specific embodiment, the microorganism is Clostridium ljungdahlii DSM 13528 (or ATCC 55383).

Nucleic acids The present invention also provides one or more nucleic acids or nucleic acid constructs used to make the recombinant microorganisms of the present invention.

  In one embodiment, the nucleic acid comprises a sequence encoding one or more enzymes of the mevalonate (MVA) pathway and optionally the DXS pathway, and is expressed in the microorganism by fermentation of a substrate comprising CO when expressed in the microorganism. The above terpenes and / or precursors thereof are produced. In one particular embodiment, the present invention comprises two or more enzymes that, when expressed in a microorganism, cause the microorganism to produce one or more terpenes and / or precursors thereof by fermentation of a substrate comprising CO. Nucleic acids encoding are provided. In one embodiment, the nucleic acids of the invention encode three, four, five or more such enzymes.

In one embodiment, the one or more enzymes encoded by the nucleic acid are from the mevalonate (MVA) pathway,
a) thiolase (EC 2.3.1.9),
b) HMG-CoA synthase (EC 2.3.3.10),
c) HMG-CoA reductase (EC 1.1.1.88),
d) Mevalonate kinase (EC 2.7.1.36),
e) phosphomevalonate kinase (EC 2.7.4.2),
f) selected from the group consisting of diphosphomevalonate decarboxylase (EC 4.1.1.33), and g) any variant thereof that is functionally equivalent.

In a further embodiment, the one or more enzymes encoded by the nucleic acid are from the DXS pathway,
a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7),
b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267),
c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60),
d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148),
e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12),
f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1),
g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2), and h) selected from the group consisting of any variant thereof that is functionally equivalent Is done.

In a further embodiment, the nucleic acid is expressed or overexpressed, an exogenous enzyme that is expressed and encodes one or more additional enzymes that result in the production of terpene compounds and / or precursors thereof, or is overexpressed. Endogenous enzymes are
a) Geranyltransferase Fps (EC: 2.5.1.10),
b) heptaprenyl diphosphate synthase (EC: 2.5.1.10),
c) Octaprenyl-diphosphate synthase (EC: 2.5.1.90),
d) isoprene synthase (EC 4.2.2.37),
e) Isopentenyl-diphosphate Δ-isomerase (EC EC 5.3.3.2)
f) Farnesene synthase selected from the group consisting of synthase (EC 4.2.3.4/EC 4.2.3.47), and g) any functionally equivalent variant thereof.

  Exemplary amino acid and nucleic acid sequences encoding each of the above enzymes are provided herein or can be obtained from GenBank as previously described herein. However, in view of the information contained in this specification, GenBank and other databases, and the genetic code, alternative nucleic acid sequences encoding these enzymes or functionally equivalent variants thereof will be apparent to those skilled in the art. It is.

  In a further embodiment, a nucleic acid encoding a thiolase (thIA) from Clostridium acetobutylicum ATCC 824 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 40 below, or a functionally equivalent variant thereof.

  In a further embodiment, the thiolase is Staphylococcus aureus subsp. A nucleic acid encoding thiolase, which is an acetyl-CoA c-acetyltransferase (vraB) derived from aureus Mu50, is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 41 below, or a functionally equivalent variant thereof. is there.

  In a further embodiment, Staphylococcus aureus subsp. The nucleic acid encoding 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) from Aureus Mu50 is encoded by the nucleic acid sequence illustrated in SEQ ID NO: 42 below, or is a functionally equivalent variant thereof .

  In a further embodiment, Staphylococcus aureus subsp. A nucleic acid encoding hydroxymethylglutaryl-CoA reductase (HMGR) derived from aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 43 below, or a functionally equivalent variant thereof.

  In a further embodiment, Staphylococcus aureus subsp. The nucleic acid encoding mevalonate kinase (MK) derived from Aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 51 below, or a functionally equivalent variant thereof.

  In a further embodiment, Staphylococcus aureus subsp. A nucleic acid encoding phosphomevalonate kinase (PMK) derived from aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 52 below, or a functionally equivalent variant thereof.

  In a further embodiment, Staphylococcus aureus subsp. A nucleic acid encoding diphosphomevalonate decarboxylase (PMD) derived from aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 53 below, or a functionally equivalent variant thereof.

  In a further embodiment, C.I. The nucleic acid encoding deoxyxylulose 5-phosphate synthase from autoethanogenum is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 1 and / or has an amino sequence exemplified in SEQ ID NO: 2 below, or functionally The equivalent variant.

  In one embodiment, the nucleic acid encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267) has the sequence of SEQ ID NO: 3, or functionally The equivalent variant.

  In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60) has the sequence of SEQ ID NO: 5, or It is a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.148) has the sequence of SEQ ID NO: 7, or functionally The equivalent variant.

  In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC: 4.6.1.12) has the sequence of SEQ ID NO: 9, Or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.17.7.1) has the sequence of SEQ ID NO: 11 Or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2) has the sequence of SEQ ID NO: 13, or functionally The equivalent variant.

  In a further embodiment, Escherichia coli str. K-12 substr. The nucleic acid encoding geranyltransferase (ispA) derived from MG1655 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 56 below, or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding heptaprenyl diphosphate synthase has the sequence of SEQ ID NO: 17, or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid encoding octaprenyl-diphosphate synthase (EC: 2.5.1.90), wherein the octaprenyl-diphosphate synthase is a polyprenyl synthetase, is encoded by SEQ ID NO: 19, or It is a functionally equivalent variant thereof.

  In one embodiment, a nucleic acid encoding an isoprene synthase (ispS) from Poplar tremuloides is exemplified in SEQ ID NO: 21 below, or a functionally equivalent variant thereof.

  In a further embodiment, the nucleic acid encoding isopentenyl-diphosphate Δ-isomerase (idi) from Clostridium beijerinckii is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 54, or is a functionally equivalent variant thereof .

  In a further embodiment, a nucleic acid encoding alpha farnesene synthase (FS) from Malus x domestica is encoded by the nucleic acid sequence illustrated in SEQ ID NO: 57 below, or a functionally equivalent variant thereof.

  In one embodiment, the nucleic acid of the invention further comprises a promoter. In one embodiment, the promoter allows constitutive expression of the gene under its control. However, inducible promoters may be used. Those skilled in the art will readily understand suitable promoters for use in the present invention. Preferably, the promoter is capable of directing high levels of expression under suitable fermentation conditions. In certain embodiments, the Wood-Ljungdahl cluster promoter is used. In another embodiment, a phosphotransacetylase / acetate kinase promoter is used. In another embodiment, a pyruvate: ferredoxin oxidoreductase promoter, an Rnf composite operon promoter or an ATP synthase operon promoter is used. In one particular embodiment, the promoter is C.I. It is derived from autoethanogenum.

  The nucleic acids of the invention may be kept off-chromosome during transformation of the parent microorganism or may be adapted to integrate into the genome of the microorganism. Thus, the nucleic acids of the invention can be used with additional nucleotide sequences adapted to support integration (eg, regions that allow homologous recombination and targeted integration into the host genome), or stable expression of extrachromosomal constructs and Additional nucleotide sequences adapted to assist replication (eg, origin of replication, promoter, and other control sequences) may be included.

  In one embodiment, the nucleic acid is a nucleic acid construct or vector. In one particular embodiment, the nucleic acid construct or vector is an expression construct or vector, although other constructs and vectors as used for cloning are also encompassed by the invention. In one particular embodiment, the expression construct or vector is a plasmid.

  The expression constructs / vectors of the invention may include any number of regulatory elements, if desired, in addition to additional genes and promoters suitable for expression of additional proteins. In one embodiment, the expression construct / vector includes one promoter. In another embodiment, the expression construct / vector includes two or more promoters. In one particular embodiment, the expression construct / vector includes one promoter for each gene to be expressed. In one embodiment, the expression construct / vector includes one or more ribosome binding sites, preferably a ribosome binding site for each gene to be expressed.

  It will be apparent to those skilled in the art that the nucleic acid sequences and construct / vector sequences described herein may include standard linker nucleotides, such as those required for ribosome binding sites and / or restriction sites. Such linker sequences are not to be construed as necessary and are not intended to limit the defined sequence.

  Nucleic acids and nucleic acid constructs, including expression constructs / vectors of the invention, may be constructed using any of the standard prior art. For example, chemical synthesis or recombinant techniques may be used. Such techniques are described, for example, by Sambrook et al. (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Further exemplary techniques are described in the Examples section herein below. In essence, individual genes and regulatory elements are operably linked to each other such that the gene is expressed to form the desired protein. Suitable vectors for use in the present invention are understood by those skilled in the art. However, by way of example, the following vectors would be suitable: pMTL80000 vector, pIMP1, pJIR750, and the plasmids exemplified in the examples herein below.

  Obviously, the nucleic acids of the invention may be in any suitable form including RNA, DNA, or cDNA.

  The present invention also provides host organisms, particularly microorganisms, including viruses, bacteria, and yeast, comprising one or more of any of the nucleic acids described herein.

Biological Production Methods One or more exogenous nucleic acids may be delivered to the parental microorganism as naked nucleic acids, or may be formulated with one or more agents to facilitate the transformation process (eg, liposome binding). Nucleic acids, organisms containing nucleic acids). The one or more nucleic acids may be DNA, RNA, or combinations thereof as appropriate. Restriction enzymes may be used in certain embodiments; see, for example, Murray, N .; E. (2000) Microbial. Molec. Biol. Rev. 64, 412. reference.

  The microorganisms of the present invention may be prepared from a parental microorganism and one or more exogenous nucleic acids using any prior art for producing recombinant microorganisms. By way of example, transformation (transformation or transfection) may be accomplished by electroporation, sonication, polyethylene glycol mediated transformation, or chemical or natural competence, or conjugation. Suitable transformation techniques are described in, for example, Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold 9 Spring Spring, 198. It is described in.

  In certain embodiments, due to the restriction system active in the transformed microorganism, the nucleic acid introduced into the microorganism must be methylated. This can be done using a variety of techniques, including those described below, and is further illustrated in the examples herein below.

By way of example, in one embodiment, the recombinant microorganism of the present invention comprises the following steps:
b) (i) introduction of the expression construct / vector described herein and (ii) a methylated construct / vector comprising a methyltransferase gene into a shuttle microorganism;
c) expression of methyltransferase;
d) isolation of one or more constructs / vectors from the shuttle microorganism;
e) produced by a method comprising introduction of one or more constructs / vectors into the target microorganism.

  In one embodiment, the methyltransferase gene of Step B is constitutively expressed. In another embodiment, the expression of the methyltransferase gene of Step B is induced.

  Shuttle microorganisms are microorganisms, preferably restriction-negative microorganisms that promote methylation of the nucleic acid sequences that make up the expression construct / vector. In certain embodiments, the shuttle microorganism is a restriction negative E. coli. E. coli, Bacillus subtilis, or Lactococcus lactis.

  The methylation construct / vector includes a nucleic acid sequence encoding a methyltransferase.

  When the expression construct / vector and the methylation construct / vector are introduced into the shuttle microorganism, the methyltransferase gene present on the methylation construct / vector is induced. In one particular embodiment of the invention, the methylation construct / vector comprises an inducible lac promoter and the addition of lactose or an analogue thereof, more preferably isopropyl-β-D-thio-galactoside (IPTG). However, the induction may be by any suitable promoter system. Other suitable promoters include the ara, tet, or T7 system. In a further embodiment of the invention, the methylation construct / vector promoter is a constitutive promoter.

  In certain embodiments, the methylation construct / vector has a replication origin specific for the identity of the shuttle microorganism, such that any gene present on the methylation construct / vector is expressed in the shuttle microorganism. Preferably, the expression construct / vector has an origin of replication specific for the identity of the target microorganism so that any gene present on the expression construct is expressed in the target microorganism.

  Expression of the methyltransferase enzyme results in methylation of the gene present on the expression construct / vector. The expression construct / vector may then be isolated from the shuttle microorganism according to any known method. By way of example, expression constructs / vectors may be isolated using the methods described in the Examples herein.

  In one particular embodiment, both constructs / vectors may be isolated together.

  The expression construct / vector may be introduced into the target microorganism using any known method. However, by way of example, the methods described in the examples below may be used. Since the expression construct / vector is methylated, the nucleic acid sequences present on the expression construct / vector can be incorporated into the target microorganism and are successfully expressed.

  It is expected that the methyltransferase gene will be introduced into the shuttle microorganism and overexpressed. Thus, in one embodiment, the resulting methyltransferase enzyme may be collected using known methods and used in vitro to methylate the expression plasmid. The expression construct / vector may then be introduced into the target microorganism for expression. In another embodiment, after the methyltransferase gene is induced in the genome of the shuttle microorganism, the expression construct / vector is introduced into the shuttle microorganism, one or more constructs / vectors are isolated from the shuttle microorganism, and then The expression construct / vector is introduced into the target microorganism.

  It is anticipated that the expression construct / vector and methylation construct / vector as defined above may be combined to provide the composition. Such compositions are particularly useful for avoiding the restriction barrier mechanism and producing the recombinant microorganisms of the present invention.

  In one particular embodiment, the expression construct / vector and / or methylation construct / vector is a plasmid.

  Many suitable methyltransferases used to produce the microorganisms of the present invention will be apparent to those skilled in the art. However, by way of example, the Bacillus subtilis phage ΦT1 methyltransferase and the methyltransferase described in the examples herein below may be used. In one embodiment, the methyltransferase has the amino acid sequence of SEQ ID NO: 60, or a functionally equivalent variant thereof. In view of the desired methyltransferase sequence and genetic code, the nucleic acid encoding the appropriate methyltransferase will be apparent. In one embodiment, the nucleic acid encoding the methyltransferase is as described in the Examples herein below (eg, the nucleic acid of SEQ ID NO: 63, or a functionally equivalent variant thereof). ).

  Any number of constructs / vectors adapted to allow expression of the methyltransferase gene may be used to generate the methylated construct / vector. However, as an example, the plasmids described in the examples below may be used.

Method of Production The present invention relates to one or more terpenes and / or precursors thereof, and optionally one or more by microbial fermentation comprising fermenting a substrate comprising CO using the recombinant microorganism of the present invention. Methods for producing other products are provided. Preferably, the one or more terpenes and / or precursors thereof are the main fermentation products. The method of the present invention may be used to reduce the total amount of atmospheric carbon emissions from industrial processes.

  Preferably, the fermentation comprises the step of anaerobically fermenting the substrate in a bioreactor using the recombinant microorganism of the present invention to produce at least one or more terpenes and / or precursors thereof.

  In one embodiment, the one or more terpenes and / or precursors thereof are mevalonic acid, IPP, dimethylallyl diphosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), And selected from farnesene.

  Instead of producing isoprene directly from the key terpenoid intermediates IPP and DMAPP and then using it to synthesize longer chain terpenes, C10 monoterpenoids or C15 sesquiterpenoids, etc. directly via geranyltransferase It is also possible to synthesize longer chain terpenes (see Table 6). Farnesene can be produced from the C15 sesquiterpenoid building block farnesyl-PP, which, like ethanol, can be used as a transportation fuel.

In one embodiment, the method comprises:
(A) providing a bioreactor comprising a culture of one or more microorganisms of the present invention with a substrate comprising CO; and (b) anaerobically fermenting the bioreactor culture to at least one terpene. And / or producing precursors thereof.

In one embodiment, the method comprises:
a) supplementing the CO-containing gas produced as a result of the industrial process;
b) comprising the step of anaerobic fermentation of a CO-containing gas to produce at least one or more terpenes and / or precursors thereof by culture comprising one or more microorganisms of the present invention.

In one embodiment of the invention, the gaseous substrate fermented by the microorganism is a gaseous substrate comprising CO. The gaseous substrate may be CO with waste gas obtained as a by-product of an industrial process, or may be from some other source, such as from automobile exhaust fumes. In some embodiments, the industrial process is selected from the group consisting of ferrous metal product manufacturing such as steelmaking, non-ferrous product manufacturing, petroleum refining process, coal vaporization, power generation, carbon black generation, ammonia generation, methanol production and coal production. Is done. In these embodiments, the CO-containing gas may be supplemented from an industrial process prior to being vented to the atmosphere using any convenient method. CO may be a component of synthesis gas (a gas containing carbon monoxide and hydrogen). CO produced from an industrial process is usually burned to produce CO _2. Accordingly, the present invention is particularly useful for producing terpenes that uses the release of CO ₂ greenhouse gas as reduced and biofuels. Depending on the composition of the gaseous CO-containing substrate, it may also be desirable to treat undesired impurities, such as dust particles, before introducing the substrate into the fermentation. For example, the gaseous substrate may be filtered or washed using known methods.

  In order to generate CO in response to bacterial growth and at least one or more terpenes and / or precursors thereof, an appropriate liquid nutrient medium needs to be supplied to the bioreactor in addition to the CO-containing substrate gas. It will be understood. The substrate and medium may be fed to the bioreactor in a continuous form, batch, or batch feed form. The nutrient medium contains enough vitamins and minerals to allow the growth of the microorganisms used. Anaerobic media suitable for fermentation to produce terpenes and / or precursors thereof using CO are known to those skilled in the art. For example, a suitable medium is described in Biebel (2001). In one embodiment of the invention, the culture medium is as described in the examples herein below.

  The fermentation is desirably performed under suitable conditions to generate CO in response to fermentation of at least one or more terpenes and / or precursors thereof. Reaction conditions to be considered include pressure, temperature, gas flow rate, liquid flow rate, medium pH, medium redox potential, stirring rate (if a continuous stirred tank reactor is used), inoculation level, in liquid phase The maximum gas substrate concentration to ensure that the CO is not limited, and the maximum product concentration to avoid product inhibition.

  Moreover, in many cases it is desirable to increase the CO concentration of the substrate stream (or CO partial pressure in the gaseous substrate), thereby increasing the efficiency of the fermentation reaction in which CO is the substrate. By operating at increased pressure, the rate of CO transfer from the gas phase to the liquid phase is significantly increased and CO is taken up by the microorganism as a carbon source for the production of at least one or more terpenes and / or precursors thereof. Can be. This also means that if the bioreactor is maintained at a pressure higher than atmospheric pressure, the retention time (defined as the liquid volume of the bioreactor divided by the input gas flow rate) can be reduced. . Optimal reaction conditions will depend in part on the particular microorganism of the invention used. However, it is generally preferred to carry out the fermentation at a pressure higher than atmospheric pressure. Also, the conversion ratio provided from CO to one or more terpenes and / or their precursors depends in part on the function of the retention time of the substrate, and it is now necessary to achieve the desired retention time. The volume of bioreactor required by the use of a pressurized system can be greatly reduced, resulting in a significant reduction in the capital cost of the fermentation apparatus. In accordance with the embodiment disclosed in US Pat. No. 5,593,886, the bioreactor capacity can be reduced in a linear ratio with increasing reactor operating pressure. That is, a bioreactor operating at 10 atmospheres requires only 1/10 the capacity of a bioreactor operating at 1 atmosphere.

  As an example, the benefits of performing a gas to ethanol fermentation at increased pressure are described. For example, WO 02/08438 performs a gas to ethanol fermentation under pressures of 30 psig and 75 psig to obtain ethanol with a productivity of 150 g / day and 369 g / day, respectively. However, in the example of fermentation carried out at atmospheric pressure using similar media and input gas composition, it was found that 10-20 times less ethanol per liter was produced per day.

  Also, the introduction rate of the CO-containing gaseous substrate is desirably such that it guarantees that the liquid phase CO concentration is not limited. This is due to the fact that one or more products can be consumed in culture as a result of the limited CO conditions.

The composition of the gas stream used to feed the fermentation reaction significantly affects the efficiency and / or cost of the reaction. For example, O ₂ reduces the efficiency of the anaerobic fermentation process. Undesirable or unnecessary gas treatment at the stage of the fermentation process before and after fermentation can increase the burden of such a stage (eg if the gas stream is compressed before entering the bioreactor) Unnecessary energy is used to compress gases that are not necessary for fermentation). Accordingly, it is desirable to treat substrate streams, particularly substrate streams from industrial sources, to remove unwanted components and increase the concentration of desirable components.

  In certain embodiments, the bacterial culture of the invention is maintained in an aqueous culture medium. Preferably, the aqueous culture medium is a minimal anaerobic bacterial growth medium. Suitable media are known to those skilled in the art and are described, for example, in US Pat. Nos. 5,173,429, 5,593,886, and WO 02/08438, and herein As described in the examples below.

  Mixed streams containing terpenes and / or their precursors, or one or more terpenes, their precursors, and / or one or more other products can be fractional distillation or evaporation, pervaporation, gas stripping And may be recovered from the fermentation broth by methods known to those skilled in the art, such as extractive fermentation including liquid-liquid extraction and the like.

  In certain preferred embodiments of the invention, the one or more terpenes and / or precursors thereof and one or more products continuously remove a portion of the broth from the bioreactor and remove microbial cells from the broth (filtered). Recovered from the fermentation broth by separating (and more conveniently) and recovering one or more products from the broth. The alcohol is easily recovered, for example, by distillation. Acetone may be recovered, for example, by distillation. Any acid produced may be recovered, for example, by adsorption onto activated carbon. The separated microbial cells are preferably returned to the fermentation bioreactor. Permeate free of cells remaining after any alcohol and acid has been removed is also preferably returned to the fermentation bioreactor. Prior to returning to the bioreactor, additional nutrients (such as vitamin B) may be added to the cell-free permeate to replenish the nutrient medium.

  Also, if the pH of the broth is adjusted as described above to enhance the absorption of acetic acid into activated carbon, the pH should be similar to that of the broth in the fermentation bioreactor before returning to the bioreactor. Should be readjusted.

Examples The invention is described in more detail with reference to the following non-limiting examples.

Example 1: C.I. for production of isoprene from CO. Expression of isoprene synthase in autoethanogenum. autoethanogenum and C.I. A terpene biosynthetic gene in carbon oxide-trophic acetic acid producing bacteria such as ljungdahlii was identified. The recombinant microorganism was modified to produce isoprene. Isoprene is naturally emitted from several plants such as poplar to protect leaves from UV radiation. The poplar isoprene synthase (EC 4.2.2.37) gene was codon-optimized to produce isoprene from CO. Introduced into autoethanogenum. This enzyme converts the intermediate DMAPP (dimethylallyl diphosphate), the key to terpenoid biosynthesis, to isoprene by an irreversible reaction (FIG. 1).

Strains and growth conditions:
All subcloning steps are performed in E. coli using standard strains and growth conditions previously described. Was performed on coli (Sambrook et al, Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989; Ausubel et al, Current protocols in molecular biology, John Wiley & Sons, Ltd., Hoboken, 1987) .

C. autoethanogenum DSM10061 and DSM23693 (DSM10061 derivative) were obtained from DSMZ (The German Collection of Microorganisms and Cell Cultures, Inoffenstrasse 7 B, 38124, City of Braunschweig, Germany). Growth was performed at 37 ° C. using strict anaerobic conditions and techniques (Hungate, 1969, Methods in Microbiology, vol. 3B. Academic Press, New York: 117-132; Wolfe, 1971, Ad. Av. Physiol., 6: 107-146). Chemically defined PETC media without yeast extract (Table 1) and 30 psi carbon monoxide with steelmaking exhaust as the sole carbon and energy source (collected at New Zealand Steel, Glenbrook, New Zealand, composition: 44 % CO, 32% N ₂ , 22% CO ₂ , 2% H ₂ )
It was used.

Construction of expression plasmid:
In the present invention, standard recombinant DNA and molecular cloning techniques were used (Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Harbourology, 198 Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current protocols in molecular biology.John Wiley & Sons, ltd., 7). Poplar tremuloides isoprene synthase (AAQ16588.1; GI: 33358229) was synthesized with optimized codons (SEQ ID NO: 21). C. The gene was expressed using the autoethanogenum pyruvate: ferredoxin oxidoreductase promoter region (SEQ ID NO: 22).

  Genomic DNA from Clostridium autoethanogenum DSM23693 was isolated using a modification of the method by Bertram and Durre (1989). A 100 ml culture grown overnight was collected (6,000 × g, 15 min, 4 ° C.), washed with potassium phosphate buffer (10 mM, pH 7.5) and washed with 1.9 ml STE buffer (50 (MmM Tris-HCl, 1 mM EDTA, 200 mM sucrose; pH 8.0). 300 μl of lysozyme (˜100,000 U) was added and the mixture was incubated for 30 minutes at 37 ° C., then 280 μl of 10% (w / v) SDS solution was added and incubated for another 10 minutes. RNA was s digested at room temperature by adding 240 μl EDTA solution (0.5 M, pH 8), 20 μl Tris-HCl (1 M, pH 7.5), and 10 μl RNase A (Fermentas Life Sciences). Thereafter, 100 μl of proteinase K (0.5 U) was added, and protein degradation was performed at 37 ° C. for 1 to 3 hours. Finally, 600 μl of sodium perchlorate (5M) was added, followed by phenol-chloroform extraction and isopropanol precipitation. Quantitative and qualitative analysis of DNA was performed by spectroscopic measurement. The promoter sequence of pyruvate: ferredoxin oxidoreductase was used as oligonucleotides Ppfor-NotI-F (SEQ ID NO: 23: AAGCGGCCGCAAAATAGTTTATATAATGC) and Ppfor-NdeI-R (SEQ ID NO: 24: TACGCATATGATCTCCTCTChCIT Bio-Rad Laboratories) and were amplified by PCR using the following program and first denaturation (98 ° C., 30 seconds) followed by 32 cycles of denaturation (98 ° C., 10 seconds), annealing (50-62 ° C. 30-120 seconds) and extension (72 ° C., 30-90 seconds), followed by a final extension step (72 ° C., 10 minutes) while).

Construction of an isoprene synthase expression plasmid:
Construction of the expression plasmid E. coli strains DH5α-T1 ^R (in vitro gen) and XL1-Blue MRF ′ Kan (Stratagene). In the first step, the amplified Ppfor promoter region was transformed into E. coli using NotI and NdeI restriction sites. The plasmid pMTL85146 was produced by cloning into the E. coli-Clostridium shuttle vector pMTL85141 (FJ797651.1; Nigel Minton, University of Nottingham; Heap et al., 2009). As a second step, ispS was cloned into pMTL85146 using restriction sites NdeI and EcoRI, resulting in plasmid pMTL85146-ispS (FIG. 2, SEQ ID NO: 25).

C. Transformation and expression in autoethanogenum Prior to transformation, as described in US Patent Publication No. 2011/0236941, C.I. autoethanogenum, C.I. ragsdalei and C.I. Using a synthesized hybrid type II methyltransferase (SEQ ID NO: 63) co-expressed on a methylated plasmid (SEQ ID NO: 64) designed from a methyltransferase gene from ljungdahlii, the DNA was transformed into E. coli. Methylated in vivo in E. coli.

  Both the expression plasmid and the methylated plasmid were treated with restriction-negative E. coli. E. coli XL1-Blue MRF 'Kan (Stratagene) was transformed into the same cells, which were compatible with their gram-(-) origin of replication (high copy ColE1 in the expression plasmid and low copy in the methylated plasmid). p15A) is possible. In vivo methylation was induced by the addition of 1 mM IPTG and the methylated plasmid was isolated using QIAGEN Plasmid Midi Kit (QIAGEN). The resulting mixture is C.I. Used for transformation experiments with autoethanogenum DSM23693, but only the high-volume (high copy) expression plasmid has a gram-(+) origin of replication (repL) allowing replication in Clostridium.

C. Transformation into autoethanogenum:
During all transformation experiments, C.I. autoethanogenum DSM23693 contains 1 g / L yeast extract and 10 g / l fructose and 30 psi steelmaking flue gas as a carbon source (collected from New Zealand Steel, New Zealand Glenbrook, composition: 44% CO, 32% N ₂ , 22% CO _2, PETC medium supplemented 2% _{H 2)} (Table 1) were grown in.

To make competent cells, 50 ml of C.I. The autoethanogenum DSM23693 culture was subcultured to fresh medium and continuously subcultured for 3 days. These cells were used to seed 50 ml PETC medium containing 40 mM DL-threonine at an OD _{600 m} of 0.05. When the OD _{600m of the} culture reached 0.4, the cells were transferred to an anaerobic chamber and collected at 4,700 xg and 4 ° C. The culture was washed twice with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl2, 7 mM sodium phosphate, pH 7.4) and finally suspended in 600 μl of fresh electroporation buffer. This mixture was transferred to a pre-ice-cooled electroporation cuvette with a 0.4 cm electrode gap containing 1 μg of methylated plasmid mixture and immediately Gene Pulser Xcell Electro at the following settings (2.5 kV, 600Ω, and 25 μF). Electricity was conducted using a poration system (Bio-Rad). A time constant of 3.7-4.0 ms was achieved. The culture was transferred to 5 ml fresh medium. Cell regeneration was monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon Spectrophotometer (Thermo) equipped with a tube holder. After the initial biomass decline, the cells began to grow again. After doubling the biomass from this point, cells are harvested and suspended in 200 μl fresh medium and selective PETC containing the appropriate antibiotic substrate 4 μg / ml clarithromycin or 15 μg / ml thianphenicol. Plates were plated (containing 1.2% Bacto ™ agarose (BD)). Colonies were visible in 4-5 days with 30 psi steelmaking gas at 37 ° C. after sowing.

  This colony was used to inoculate 2 ml of PETC medium with antibiotic substrate. After growth occurred, the culture was scaled up to a volume of 5 ml and then a volume of 50 ml with 30 psi steelmaking gas as the sole carbon source.

Confirmation of successful transformation:
In order to verify DNA transfer, plasmid minipreps were performed from a 10 ml culture volume using a Zppy plasmid miniprep kit (Zymo). Since the quality of the isolated plasmid was not sufficient for restriction digestion due to the exonuclease activity of Clostridium [Burchhardt and Durre, 1990], the oligonucleotide pair colE1-F (SEQ ID NO: 65: CGTCAGACCCCGTAGAA) + colE1-R ( PCR was performed on the isolated plasmid using SEQ ID NO: 66: CTCTCCTGTTTCCGACCCT). PCR was performed using the iNtRON Maximum Premix PCR kit (Intron Bio Technologies) under the following conditions: first denaturation (94 ° C., 2 min), followed by 35 cycles of denaturation (94 ° C., 20 sec), annealing. (55 ° C., 20 seconds), and extension (72 ° C., 60 seconds), followed by a final extension step (72 ° C., 5 minutes).

  To confirm the identity of the clone, the DNA genome is Isolated from a 50 ml culture of autoethanogenum DSM23693 (see above). Oligonucleotides (SEQ ID NO: 67: CCGAATTCGTCGACAACAGAGTTTGATCCCTGCTCAG) and rP2 (SEQ ID NO: 68: CCCGGGATCCAAGCTTACGGCACTCTGTTACGACTTT) [Weisberg et al. , 1991] and iNtRON Maximum Premix PCR kit (Intron Bio Technologies), PCR for 16s rRNA was performed under the following conditions: initial denaturation (94 ° C., 2 min) followed by 35 cycles of denaturation ( 94 ° C., 20 seconds), annealing (55 ° C., 20 seconds), and extension (72 ° C., 60 seconds), followed by final extension step (72 ° C., 5 minutes). The result of the sequence is C.I. It was at least 99.9% identical to the autoethanogenum 16s rRNA gene (rrsA) (Y18178, GI: 7271109).

Expression of isoprene synthase gene In qRT-PCR experiments, the introduced isoprene synthase gene was expressed in C.I. This was carried out to confirm that it was successfully expressed in autoethanogenum.

Cultures containing isoprene synthase plasmid pMTL 85146-ispS and control cultures without plasmid were collected in a 50 ml serum bottle and 30 psi steelmaking exhaust (New Zealand Steel, Glenbrook, New Zealand, as the sole source of energy and carbon, Composition: 44% CO, 32% N ₂ , 22% CO ₂ , 2% H ₂ ) was used to grow in PETC medium (Table 1). A 0.8 ml sample was taken during a logarithmic growth phase of OD _{600 nm} around 0.5 and mixed with 1.6 ml RNA protection reagent (Qiagen). This mixture was centrifuged (6,000 × g, 5 min, 4 ° C.) and the cell pellet was quickly frozen in liquid nitrogen and stored at −80 ° C. until RNA extraction. Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to manual protocol 5. Cell disruption was performed by passing the mixture 10 times through a syringe and eluting with 50 μl RNase / DNase free water. After DNase I treatment using DNA-free® Kit (Ambion), a reverse transcription step was performed using SuperScript III Reverse Transscriptase Kit (in vitro gen, Carlsbad, CA, USA). RNA was examined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), Qubit Fluorometer (in vitro gen, Carlsbad, CA, USA) and by gel electrophoresis. Non-RT controls were performed for each oligonucleotide pair. All qRT-PCR reactions include 25 ng cDNA template, 67 nM of each oligonucleotide (Table 2), and 1x iQ ™ SYBR ™ Green Supermix (Bio-Rad Laboratories, Carlsbad, CA, USA) Duplicates were performed using MyiQ ™ Single Color Detection System (Bio-Rad Laboratories, Carlsbad, Calif., USA) in a total reaction volume of 15 μl. The reaction conditions were 95 ° C. for 3 minutes, followed by 40 cycles of 95 ° C. for 15 seconds, 55 ° C. for 15 seconds and 72 ° C. for 30 seconds. For detection of oligonucleotide dimerization or other amplification byproducts, melting curve analysis was performed immediately after completion of qPCR (38 cycles from 58 ° C. to 95 ° C. at 1 ° C./s). Two housekeeping genes (guanylate kinase and formate tetrahydrofolate ligase) were included in each cDNA sample for normalization. Relative gene expression determination was performed using the Relative Expression Software Tool (REST (c)) 2008 V2.0.7 (38). A standard curve was generated using serial dilutions of cDNA over 4 log units, and the resulting amplification efficiency was used to calculate the concentration of mRNA.

  No amplification was observed in the wild type strain using the oligonucleotide pair ispS, but a signal was measured using the ispS oligonucleotide pair in the strain with the plasmid pMTL 85146-ispS, indicating that the ispS gene is well expressed. confirmed.

Example 2-Expression of isopentenyl-diphosphate Δ-isomerase for conversion between key terpene precursors DMAPP (dimethylallyl diphosphate) and IPP (isopentenyl diphosphate) Precursor DMAPP (dimethylallyl diphosphate) The availability and balance of phosphoric acid) and IPP (isopentenyl diphosphate) is important for the production of terpenes. The DXS pathway synthesizes IPP and DMAPP equally, whereas the mevalonate pathway produces only IPP. Production of isoprene requires only the precursor DMAPP present with isoprene synthase, while production of higher terpenes and terpenoids requires an equal amount of IPP available to produce geranyl-PP by geranyl transferase. And having DMAPP.

Construction of isopentenyl-diphosphate Δ-isomerase expression plasmid:
C. encoding isopentenyl-diphosphate Δ-isomerase (YP — 001310174.1) The isopentenyl-diphosphate Δ-isomerase gene idi (Gene ID: 5294264) from beijerinckii was cloned downstream of ispS. This gene was designated as C.I. C. obtained using the same method described above with autoethanogenum. Beijerckii NCIMB8052 genomic DNA-derived oligonucleotides Idi-Cbei-SacI-F (SEQ ID NO: 26: GTGAGCTCGAAAGGGGAAATTAATG) and Idi-Cbei-KpnI-R (SEQ ID NO: 27: ATGGTACCCCAAATCTTTGTA) were used. This PCR product was cloned into the vector pMTL 85146-ispS using SacI and KpnI restriction sites, resulting in plasmid pMTL85146-ispS-idi (SEQ ID NO: 28). The anaerobic resistance marker was exchanged from catP to ermB (vector pMTL82254 (FJ79766.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using plasmids P, 85S, using the restriction enzymes PmeI and FseI. -Idi was formed (FIG. 3).

C. Transformation and expression with autoethanogenum was performed as described for plasmid pMTL 85146-ispS. After successful transformation, growth experiments were performed on 50 ml serum bottles and steelmaking exhaust as the sole energy and carbon source (collected at New Zealand Steel, New Zealand Glenbrook, composition: 44% CO, 32% N ₂ , 22% CO _2. 2% H ₂ ) was used in PETC medium (Table 1). To confirm that the plasmid was successfully introduced, plasmid miniprep DNA was run from the transformants as described above. colE1 (colE1-F: SEQ ID NO: 65: CGTCAGACCCCGTTAGAAA and colE1-R: SEQ ID NO: 66: CTCTCCTGTTCCCACCCCCT), ermB (ermB-F: SEQ ID NO: 106: TTTGTATAATTAAGAAGAGAG and ermB-R: SEQ ID NO: 107: GTTAGAC PCR on an isolated plasmid using an oligonucleotide pair targeting idi (Idi-Cbei-SacI-F: SEQ ID NO: 26: GTGAGCTCCGAAAGGGGAAAATATG and Idi-Cbei-KpnI-R: SEQ ID NO: 27: ATGGTACCCCAAATTCTTTTATAGCG) Successful transformation was confirmed (FIG. 8). Similarly, 16s rRNA amplicons obtained by extracting genomic DNA from these transformations and using oligonucleotides fD1 and rP2 (see above) were obtained from C.I. It was confirmed to be 99.9% identical to the gene for 16S rRNA of autoethanogenum (Y18178, GI: 7271109).

  Oligonucleotide pair for isopentenyl-diphosphate Δ-isomerase gene idi (idi-F, SEQ ID NO: 71: ATA CGT GCT GTA GTC ATC CAA GAT A and idiR, SEQ ID NO: 72: TCT TCA AGT TCA CAT GTA AAA CCC A) and C. aureus with plasmid pMTL 85146-ispS-idi. Using samples from growth experiments in serum bottles using autoethanogenum, confirmation of successful gene expression was performed as described above. A signal for the isoprene synthase gene ispS was also observed (FIG. 14).

Example 3-Overexpression of the DXS pathway To improve the flow through the DXS pathway, genes in the pathway were overexpressed. The first step of the pathway, the conversion of pyruvate and D-glyceraldehyde-3-phosphate (G3P) to deoxyxylulose 5-phosphate (DXP / DXPS / DOXP), is deoxyxylulose 5-phosphorus. Catalyzed by acid synthase (DXS).

Construction of a DXS overexpression expression plasmid:
C. The autoxanthogenum dxs gene was converted to oligonucleotides Dxs-SalI-F (SEQ ID NO: 29: GCAGTCCGACTTTATATAAGGGATAGATAA) and Dxs-XhoI-R (SEQ ID NO: 30: TGCTCGAGTTAAATATGTAG) Amplified from genomic DNA. The amplified gene was then cloned into plasmid pMTL85246-ispS-idi with SalI and XhoI to produce plasmid pMTL85246-ispS-idi-dxs (SEQ ID NO: 31 and FIG. 4). Sequencing of DNA using the oligonucleotides disclosed in Table 3 confirmed that ispS, idi, and dxs were successfully cloned without mutation (FIG. 5). The ispS and idi genes are described in Example 1 and Example 2, respectively.

C. B. Transformation and expression in autoethanogenum Transformation and expression with autoethanogenum was performed as described in plasmid pMTL 85146-ispS. After successful transformation, growth experiments were performed in 50 ml serum bottles and 30 psi steelmaking flue gas as a single energy and carbon source (collected at New Zealand Steel, New Zealand Glenbrook, composition: 44% CO, 32% N ₂ was performed in PETC medium (Table 1) using _{H 2) 22% CO 2,} 2%. Confirmation of gene expression was performed as described above from samples collected at OD _600nm = 0.75. Oligonucleotide pair dxs-F (SEQ ID NO: 73: ACAAAGTATCTAAGACAGGAGGGTCA) and dxs-R (SEQ ID NO: 74: GATGTCCCCACATCCCATATAAGTT)) gene dxs in wild type strains and strains with plasmid pMTL 85146-ispS-idi-dxs The expression of was measured. It was found that the mRNA level in the strain with the plasmid increased more than 3 times compared to the wild type (FIG. 15). Biomass was normalized before RNA extraction.

Example 4 Introduction and Expression of the Mevalonate Pathway The first step of the mevalonate pathway (FIG. 7) is catalyzed by a thiolase that converts two molecules of acetyl CoA to acetoacetyl CoA (and HS-CoA). This enzyme has been obtained by the same inventors, Clostridium autoethanogenum and C.I. It is successfully expressed in ljungdahlii (US Patent Publication No. 2011/0236941). The remaining gene constructs of the mevalonate pathway have been designed.

Construction of mevalonic acid expression plasmid:
Standard recombinant DNA and molecular cloning techniques were used (Sambrook, J., and Russell, D., Molecular cloning: A Laboratory Manual 3rd Ed., Cold Spring Harbor Lab Press, Cold Spring, Cold Spring 200, Cold Spring, Cold Spring 200, Cold Spring, Cold Spring 200, Cold Spring, Cold Spring, Cold Spring, H Three genes required for the synthesis of mevalonate by the first half of the mevalonate pathway, namely thiolase (thlA / vrA), HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR), are operons (P _ptaack − thlA / vrB-HMGS- _Patp- HMGR).

C. The autoethanogenum phosphotransacetylase / acetate kinase operon promoter (P _pta-ack ) (SEQ ID NO: 61) was used for the expression of thiolase and HMG-CoA synthase, while C.I. The promoter region (P _atp ) of the _{autoethanogenum} ATP synthase was used for expression of HMG-CoA reductase. Two variants of thiolase, thlA from Clostridium acetobutyricum and vraB from Staphylococcus aureu, were synthesized and flanked by NdeI and EcoRI restriction sites for further subcloning. Both HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR) were synthesized from Staphylococcus aureus and flanked on EcoRI-SacI and KpnI-XbaI restriction sites, respectively, for further subcloning. All optimized DNAs used are shown in Table 4.

Using ATP synthase promoter (P _atp ) with hydroxymethylglutaryl-CoA reductase (HMGR), oligonucleotides pUC57-F (SEQ ID NO: 46: AGCAGAGTGTACTGAGAGTGC) and pUC57-R (SEQ ID NO: 47: ACAGCTATGACCATGATTTACG), and Amplification was performed using pUC57-Patp-HMGR as a template. The 2033 bp amplified fragment was digested with SacI and XbaI and E. coli. E. coli-Clostridium shuttle vector pMTL 82151 (FJ7976; Nigel Minton, University of Nottingham, UK; Heap et al., 2009, J Microbiol Methods 79-P 79: M 79-P 79: M : 76) was obtained.

  3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) was synthesized using oligonucleotides EcoRI-HMGS_F (SEQ ID NO: 77: AGCCGTGAATTCGAGGCTTTTACTAAAACA) and EcoRI-HMGS_R (SEQ ID NO: 78: AGGCGTCTAGATGTTATCATCT) Amplified from the plasmid pGH-seq3.2. The 1391 bp amplified fragment was digested with SacI and EcoRI and ligated to the previously generated plasmid pMTL 82151-Patp-HMGR to yield pMTL 82151-HMGS-Patp-HMGR (SEQ ID NO: 79). The plasmids pMTL 82151-HMGS-Patp-HMGR (SEQ ID NO: 79) and the 1768 bp codon optimized operon Pptaack-thlA / vraB were both cut with NotI and EcoRI. A ligation reaction is performed and then E. coli. E. coli XL1-Blue MRF ′ Kan was transformed into plasmid pMTL8215-Pptaack-thlA / vrB-HMGS-Patp-HMGR (SEQ ID NO: 50).

  Five genes that are required for the synthesis of key terpenoid intermediates from mevalonate via the second half of the mevalonate pathway: mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate de Carboxylase (PMD), isopentenyl-diphosphate Δ-isomerase (idi) and isoprene synthase (ispS) were codon optimized by ATG: Biosynthetics GmbH (Merzhausen, Germany). Mevalonate kinase (MK), phosphomevalonate kinase (PMK) and diphosphomevalonate decarboxylase (PMD) were obtained from Staphylococcus aureus.

C. The promoter region (P _rnf ) (SEQ ID NO: 62) of the _RNF complex of _{autoethanogenum} is used for expression of mevalonate kinase (MK), phosphomevalonate kinase (PMK) and diphosphomevalonate decarboxylase (PMD), while C.I. The autoethanogenum pyruvate: ferredoxin oxidoreductase promoter region (P _for ) (SEQ ID NO: 22) was used for the expression of isopentenyl-diphosphate Δ-isomerase (idi) and isoprene synthase (ispS). All DNA sequences used are shown in Table 5. Codon-optimized Prnf-MK was converted into oligonucleotides NotI-XbaI-Prnf-MK_F (SEQ ID NO: 80: ATGCCGCGCCCGTAGGTCTAGATAAAAAAAATATAATATACAG) and SalI-Prnf-MGTA -Amplified from PMK-PMD. Thereafter, the amplified gene was cloned into plasmid pMTL83145 (SEQ ID NO: 49) with NotI and SalI to produce plasmid pMTL8314-Prnf-MK (SEQ ID NO: 82). The resulting plasmid and codon optimized 2165 bp fragment PMK-PMD was digested with SalI and HindIII. Ligation reaction was performed to obtain plasmid pMTL 8314-Prnf-MK-PMK-PMD (SEQ ID NO: 83).

An isoprene expression plasmid without the mevalonate pathway was obtained from the farnesene plasmid pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi, in which the isoprene synthase (ispS) located flanking the restriction sites Agel and Nhel was previously created. -Plasmid pMTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispS (SEQ ID NO: 84) was prepared by ligating to -ispA-FS (SEQ ID NO: 91). The final isoprene expression plasmid pMTL 8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispS (SEQ ID NO: 58, FIG. 10) was used using NotI and XbaI. A 4360 bp fragment of Pptaack-thlA-HMGS-Patp-HMGR derived from pMTL8215-Pptaack-thlA-HMGS-Patp-HMGR (SEQ ID NO: 50) and pMTL 8314-Prnf-MK-PMK-PMD-Pfor-i It was created by linking SEQ ID NO: 84).

Example 5-C. for production of farnesene from CO via the mevalonate pathway Incorporation of farnesene synthase into autoethanogenum Key intermediates of terpenoids Instead of producing isoprene directly from IPP and DMAPP to synthesize longer chain terpenes, direct C10 monoterpenoids or C15 via geranyltransferase It is also possible to synthesize long chain terpenes such as sesquiterpenoids (see Table 6). Farnesene can be produced from the C15 sesquiterpenoid building block farnesyl PP, which, like ethanol, can be used as a transportation fuel.

Construction of Farnesene Expression Plasmid Two genes required for synthesis of farnesene from IPP and DMAPP via the mevalonate pathway, geranyltransferase (ispA) and α-farnesene synthase (FS), optimized codons did. Geranyl transferase (ispA) is obtained from Escherichia coli str. MG1655 alphadarfarnesene synthase (FS) obtained from K-12 substr was obtained from Malus x domestica. All DNA sequences used are listed in Table 6. The codon-optimized idi was mevalonate pathway idi_F (SEQ ID NO: 86: AGGCACTCGGATAGGGCAGGATATATAATAGCAGTAG) and idi_R2 (SEQ ID NO: 87: AGGCGCCAAGCTTGGCGCCAGTGTATATTCTTT TTTCpGC). The amplified gene was then cloned into plasmid pMTL83245-Pfor with XhoI and HindIII, producing plasmid pMTL83245-Pfor-idi (SEQ ID NO: 88). The resulting plasmid and the 1754 bp codon optimized fragment of farnesene synthase were digested with HindIII and NheI. Ligation reaction was performed to obtain plasmid pMTL83245-Pfor-idi-FS (SEQ ID NO: 89). The ispA 946 bp fragment and pMTL83245-Pfor-idi-FS were digested with AgeI and HindIII and ligated, resulting in the plasmid pMTL83245-Pfor-idi-ispA-FS (SEQ ID NO: 90). A farnesene expression plasmid without an upstream mevalonate pathway links a 2516 bp fragment of Pfor-idi-ispA-FS from pMTL83245-Pfor-idi-ispA-FS to pMTL 8314-Prnf-MK-PMK-PMD Thus, plasmid pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 91) was obtained. The final farnesene expression plasmid MTL83145-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 59 and FIG. 18) uses the restriction sites NotI and XbaI. The 4630 bp fragment of Pptaack-thlA-HMGS-Patp-HMGR derived from pMTL8215-Pptaack-thlA-HMGS-Patp-HMGR (SEQ ID NO: 50) It is created by linking with FS (SEQ ID NO: 91).

C. Transformation into autoethanogenum C.I. Transformation and expression with autoethanogenum was performed as described in Example 1.

Confirmation of successful transformation The presence of MTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 59) was confirmed by the presence of a large portion of the cat gene on a series of garm + ve perplicon and a series of plasmids of pMTL831xxx. It was confirmed by colony PCR using oligonucleotides repHF (SEQ ID NO: 92: AAGAAGGGCGTATATGAAAAACTTTGT) and catR (SEQ ID NO: 93: TTCGTTTACAAAACGGCAAATGTGA) that selectively amplify the portion. A 1854 bp band was obtained (FIG. 16).

C. Expression of the downstream mevalonate pathway in autoethanogenum Mevalonate kinase (MK SEQ ID NO: 51), phosphomevalonate kinase (PMK SEQ ID NO: 52), diphosphomevalonate decarboxylase (PMD SEQ ID NO: 53), which are downstream mevalonate pathway genes Confirmation of the expression of isopentyl-diphosphate Δ-isomerase (idi; SEQ ID NO: 54), geranyl transferase (ispA; SEQ ID NO: 56) and farnesene synthase (FS SEQ ID NO: 57) is shown in Example 1. Performed as described above. The oligonucleotides used are listed in Table 7.

Rt-PCR data confirming the expression of all genes in the downstream mevalonate pathway is shown in FIG. 18, and this data is also summarized in Table 8.

Production of α-farnesene from mevalonic acid After confirming successful transformation of plasmid pMTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS, 30 psi Real Mill Gas ( Collected on New Zealand Steel, New Zealand Glenbrook, composition: 44% CO, 32% N ₂ , 22% CO ₂ , 2% H ₂ ) using 50 ml PETC medium (Table 1) in 250 ml serum bottles. went. All cultures were incubated at 37 ° C. on an orbital shaker adapted to hold serum bottles. Transformants were grown to an OD600 of ~ 0.4 before being subcultured into fresh medium supplemented with 1 mM mevalonic acid. A control without mevalonic acid was set at the same time from the same culture. GC-MS (Gas Chromatography-Mass Spectrometry) samples were taken at each time point. FIG. 17 shows representative growth curves of two control cultures and two cultures fed with 1 mM mevalonic acid. Farnesene was detected in samples taken 66 and 90 hours after the start of the experiment (FIGS. 19-21).

Detection of α-farnesene by gas chromatography-mass spectrometry For GC-MS detection of α-farnesene, in 5 ml of culture, add 2 ml of hexane and shake vigorously in a sealed glass bulk tube. Hexane extraction was performed by mixing. The tube was then incubated in a sonicated water bath for 5 minutes to facilitate phase separation. 400 μl of hexane extract was transferred to a GC vial and loaded into an autoloader. Samples were prepared using EC-1000 columns (Grace Davidson, OR, USA), VARIAN MS working (Varian Inc, CA, USA. Now Agilent Technologies 2.0) and NIST MS Engineering 2.0 (National Institute of Technology 2.0). , USA) with a VARIAN GC3800 MS4000 iontrap GC / MS (Varian Inc, CA, USA. Now Agilent Technologies). The injection volume was 1 μl and the flow rate of helium carrier gas was 1 ml / min.

  The present invention is as described herein, which is intended to enable the practice of the present invention by reading the specification without undue experimentation. However, it will be apparent to those skilled in the art that many components and parameters may be varied or modified to a specific content and changed to known equivalents without departing from the scope of the invention. is there. It is also apparent that such modifications and equivalents are incorporated herein to be set individually. The names, titles, etc. of the invention are provided to enhance the understanding of the reader of this document and are not read to limit the scope of the invention.

  If present, the entire disclosures of all patent applications, patents, and publications cited above and below are hereby incorporated by reference. However, references to any patent application, patent, and publication are not recognized and imply that they constitute valid prior art or form part of general knowledge common to any country in the world. Is not to be done.

  Unless stated otherwise, terms such as “comprise”, “comprising” and the like are to be understood as a comprehensive meaning opposite to the exclusive meaning, ie “not limiting” , Including ".

Claims (31)

An isolated, genetically modified, oxytrophic, acetic acid producing bacterium comprising an exogenous nucleic acid encoding an enzyme of the mevalonate pathway or the DXS pathway or the terpene biosynthetic pathway, whereby the bacterium Expressing an enzyme, said enzyme
a) thiolase (EC 2.3.1.9);
b) HMG-CoA synthase (EC 2.3.3.10);
c) HMG-CoA reductase (EC 1.1.1.88);
d) Mevalonate kinase (EC 2.7.1.36);
e) Phosphomevalonate kinase (EC 2.7.4.2);
f) Diphosphomevalonate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7);
g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267);
h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60);
i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148);
j) 2-C-methyl-D-erythritol 2; 4-cyclodiphosphate synthase IspF (EC: 4.6.1.12);
k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1);
l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2); geranyl transferase Fps (EC: 2.5.1.10);
m) heptaprenyl diphosphate synthase (EC: 2.5.1.10);
n) Octaprenyl-diphosphate synthase (EC: 2.5.1.90);
o) Isoprene synthase (EC 4.2.27);
p) Isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2); and q) Farnesene synthase (EC 4.2.3.4/EC 4.2.3.4)
Selected from the group consisting of
Bacteria.   2. The bacterium of claim 1, wherein the bacterium does not express an enzyme in the absence of the nucleic acid.   The bacterium according to claim 1, which expresses an enzyme under anaerobic conditions. A plasmid capable of replicating in a carbon-oxytrophic acetic acid producing bacterium comprising a nucleic acid encoding an enzyme of the mevalonate pathway or the DXS pathway or the terpene biosynthetic pathway, whereby the enzyme is transformed by the bacterium when the plasmid is in the bacterium Expressed and the enzyme is
Thiolase (EC 2.3.1.9);
a) HMG-CoA synthase (EC 2.3.3.10);
b) HMG-CoA reductase (EC 1.1.1.88);
c) Mevalonate kinase (EC 2.7.1.36);
d) Phosphomevalonate kinase (EC 2.7.4.2);
e) Diphosphomevalonate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC: 2.2.1.7);
f) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC: 1.1.1.1267);
g) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC: 2.7.7.60);
h) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC: 2.7.1.1148);
i) 2-C-methyl-D-erythritol 2; 4-cyclodiphosphate synthase IspF (EC: 4.6.1.12);
j) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC: 1.7.7.7.1);
k) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC: 1.17.1.2); geranyltransferase Fps (EC: 2.5.1.10);
l) Heptaprenyl diphosphate synthase (EC: 2.5.1.10);
m) Octaprenyl-diphosphate synthase (EC: 2.5.1.90);
n) Isoprene synthase (EC 4.2.27);
o) Isopentenyl-diphosphate Δ-isomerase (EC 5.3.3.2); and p) Farnesene synthase (EC 4.2.346 / EC 4.2.3.4)
Selected from the group consisting of
Plasmid. Converting CO and / or CO ₂ into isoprene, comprising:
Bacteria convert CO and / or CO ₂ to isoprene by passing the gaseous CO-containing substrate and / or CO ₂ -containing substrate through a bioreactor containing a culture of oxytrophic acetic acid producing bacteria in the medium. And isoprene recovery from the bioreactor,
The oxytrophic acetic acid producing bacterium is genetically modified to express isoprene synthase,
Process. An isolated, genetically modified, oxytrophic, acetic acid producing bacterium comprising a nucleic acid encoding isoprene synthase,
Thereby bacteria can express isoprene synthase and bacteria can convert dimethylallyl diphosphate to isoprene,
Bacteria.   7. The isolated, genetically modified, carbon oxide-trophic, acetic acid producing bacterium of claim 6, wherein the isoprene synthase is a Populus tremuloides enzyme.   7. The isolated, genetically modified, carbon oxytrophic, acetic acid producing bacterium of claim 6, wherein the nucleic acid is an optimized codon.   7. The isolated, genetically modified, carbon oxidative, acetic acid production of isoprene synthase under transcriptional control of a promoter of a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum Bacteria. Converting CO and / or CO ₂ to isopentenyl diphosphate (IPP), comprising:
By passing the gaseous CO-containing substrate and / or CO ₂ containing substrate in bioreactors containing a culture of carbon oxides nutritive acid producing bacteria in the culture medium, isopentenyl diphosphate bacteria CO and / or CO ₂ Converting to acid (IPP) and recovering IPP from the bioreactor,
The oxytrophic acetic acid producing bacterium has been genetically modified to express isopentyl diphosphate delta isomerase,
Process. An isolated, genetically modified, oxytrophic, acetic acid producing bacterium comprising a nucleic acid encoding isopentyl diphosphate delta isomerase comprising:
Thereby bacteria can express isopentyl diphosphate delta isomerase and bacteria can convert dimethylallyl diphosphate to isopentyl diphosphate,
Bacteria.   12. The isolated, genetically modified, oxytrophic, acetic acid producing bacterium of claim 11, wherein the nucleic acid encodes Clostridium beijerinckii isopentyl diphosphate delta isomerase.   12. The isolated, genetically modified, carbon oxide-trophic, acetic acid-producing bacterium according to claim 11, wherein the nucleic acid is under the transcriptional control of the promoter of the pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum.   12. The isolated and genetically modified of claim 11, wherein the nucleic acid is under transcriptional control of a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum and downstream of a second nucleic acid encoding isoprene synthase. A carbon-nutrient, acetic acid-producing bacterium. Converting CO and / or CO ₂ into isopentyl diphosphate (IPP) and / or isoprene,
By passing the gaseous CO-containing substrate and / or CO ₂ -containing substrate through a bioreactor containing a culture of oxytrophic acetic acid producing bacteria in the medium, the bacteria remove CO and / or CO ₂ with isopentyl diphosphate. Converting to (IPP) and / or isoprene, and recovering IPP and / or isoprene from the bioreactor,
Carbon-oxidizing nutrient acetic acid producing bacteria have been genetically modified to have an increased copy number of nucleic acid encoding deoxyxylulose 5-phosphate synthase (DXS) enzyme, the increased copy number being greater than 1 per genome ,
Process.   An isolated, genetically modified, oxytrophic, acetic acid producing bacterium comprising a copy number greater than 1 per genome of nucleic acid encoding the enzyme deoxyxylulose 5-phosphate synthase (DXS).   17. The isolated, genetically modified, oxytrophic, acetic acid producing bacterium of claim 16, further comprising a nucleic acid encoding isoprene synthase.   17. The generally modified isolated oxytrophic acetic acid-producing bacterium according to claim 16, further comprising a nucleic acid encoding isopentyl diphosphate delta isomerase.   17. The isolated, genetically modified, oxytrophic, acetic acid producing bacterium of claim 16, further comprising a nucleic acid encoding isopentyl diphosphate delta isomerase and a nucleic acid encoding isoprene synthase.   An isolated, genetically modified, oxytrophic, acetic acid producing bacterium comprising a nucleic acid encoding phosphomevalonate kinase (PMK), whereby the bacterium expresses the encoded enzyme, Bacteria that are not natural to bacteria.   21. The isolated, genetically modified carbon oxide-trophic, acetic acid-producing bacterium of claim 20, wherein the enzyme is a Staphylococcus aureus enzyme.   One or more enzymes with C.I. 21. The isolated, genetically modified, carbon oxide-trophic, acetic acid-producing bacterium of claim 20, expressed under the control of an autoethanogenum promoter.   21. The genetically modified of claim 20, further comprising a nucleic acid encoding a thiolase (thlA / vraB), a nucleic acid encoding an HMG-CoA synthase (HMGS), and a nucleic acid encoding an HMG-CoA reductase (HMGR). It is an acetic acid-producing bacterium that is nutritious for carbon oxide.   24. The isolated, genetically modified, oxytrophic, acetic acid producing bacterium of claim 23, wherein the thiolase is a Clostridium acetobutylic thiolase.   21. The isolated, genetically modified, oxytrophic, acetic acid producing bacterium of claim 20, further comprising a nucleic acid encoding diphosphomevalonate decarboxylase (PMD).   An isolated, genetically modified, carbon oxide-trophic, acetic acid producing bacterium comprising an exogenous nucleic acid encoding alpha-farnesene synthase.   The nucleic acid is C.I. 27. The isolated, genetically modified, carbon oxide-trophic, acetic acid producing bacterium of claim 26, wherein the codons are optimized for expression in an autoethanogenum.   27. The bacterium of claim 26, wherein the alpha farnesene synthase is Malus x domestica alpha farnesene synthase.   27. The isolated, genetically modified, oxytrophic, acetic acid producing bacterium of claim 26, further comprising a nucleic acid segment encoding geranyl transferase.   Geranyltransferase has been identified by E. coli. 30. The isolated, genetically modified, carbon oxide-trophic, acetic acid-producing bacterium of claim 29, which is an E. coli geranyltransferase.   Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella t from the group ermoacetica, Moorella thermotrophica, Sporomusa ovata, Sporomusa sylvaetica, Sporomusa sphaeroides, Oxobacter pfenigii, and thermoter 20 Modified, carbon oxide-trophic, acetic acid-producing bacterium.

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